Ultra pure hemoglobin solutions and blood-substitutes

ABSTRACT

A blood substitute and plasma expander comprising a cross-linked, substantially endotoxin-free hemoglobin solution and process for preparing same. The process comprises fractionating whole blood, separating out a stromal-free, sterile hemoglobin solution, chromatographically separating endotoxins from said hemoglobin solution and crosslinking the resulting endotoxin-free hemoglobin solution.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of Ser. No. 07/119,121, filed Nov. 10, 1987, nowU.S. Pat. No. 5,084,558, which is a continuation-in-part of Ser. No.07/107,421, filed Oct. 13, 1987, now abandoned, which is acontinuation-in-part of Ser. No. 06/928,345, filed Nov. 10, 1986, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for producing a novel semi-syntheticblood substitute and the novel semi-synthetic blood substitute resultingtherefrom. The novel semi-synthetic blood substitute is a hemoglobinpreparation characterized by its purity, its exceptionally low levels ofendotoxin, the absence of non-hemoglobin proteins, and its molecularcross-linking profile. The semi-synthetic blood substitute has no toxicactivity when used in a substitute fashion and possesses the property ofreversibly binding gaseous ligands such as oxygen and is useful fortransporting and supplying oxygen to vital tissues and organs.Additionally, the blood substitute serves as a blood plasma expander formanagement of disease and for maintaining circulatory integrity. Afurther aspect of the invention is the preursor or intermediate, thesubstantially pure, phospholipid-free, endotoxin-free hemoglobinsolution in uncross-linked form.

2. Description of Background Materials

Complex multicellular organisms are equipped with specialized tissueswhich are concerned with the processes of nutrition and excretion. It isthe primary function of blood to provide a link between various organsand cells of the body. Blood, red cells, plasma and other componentsmaintain a constant cellular environment by circulating through everytissue and continuously delivering nutrients to the tissues and removingwaste products and various tissues which are concerned with the tissuesecretions from them. PHYSIOLOGY, Third Edition, Edited by Edward E.Selkurt, Page 223 (1971). Blood is a viscous fluid composed of cells andplasma. More than 99% of the cells are red blood cells. The majorfunction of red blood cells is to transport hemoglobin, which in turncarries oxygen from the lungs to the tissues and CO₂ from the tissues tothe lungs. Normal red blood cells contain approximately 34 grams ofhemoglobin per 100 ml of cells. Each gram of hemoglobin is capable ofcombining with approximately 1.33 ml of oxygen. See Guyton, A. C., BASICHUMAN PHYSIOLOGY: NORMAL FUNCTION IN MECHANISMS OF DISEASE, Pages 84-85(1971).

Because of the critical and ongoing need for a therapeutic agent usefulas a blood substitute for carrying and supplying oxygen and as a bloodplasma expander, intense research efforts have been directed to thedevelopment of an adequate blood substitute. The need for a bloodsubstitute exists for replacing blood lost by acute hemorrhage, bloodlosses occuring during surgical operations, resuscitation proceduresafter acidental blood loss, and the like. Further, as a plasma expander,a blood substitute serves as a therapeutic to treat volume deficiencyshock, as an alleviant in anaphylactic and allergic shock, and forreplacing plasma lost after burns and as a result of severe diarrhea.

Hemoglobin in solution has the capability to transport oxygen and,theoretically, could be used as a substitute for red blood cells.Because hemoglobin solutions are oncotically active, these solutionsalso expand plasma volume, thereby providing a function as a plasmaexpander as well. Thus the ability to be oncotically active andtransport oxygen suggests that hemoglobin solutions would be desirablefor a resuscitation fluid where rapid initial treatment of hypovolemiaand tissue hypoxia is required. However, in order to function as anadequate resuscitation fluid, hemoglobin solutions must be capable ofmaintaining tissue oxygenation for specified periods of time.

Hemoglobin present in the blood of mammals has a fundamental property insolution of reversible oxygenation. In its natural form, mammalianhemoglobin is a conjugated, non-crosslinked protein having a molecularweight of approximately 68,000 and structurally comprised of two pairsof sub-units. Each sub-unit contains a heme group and a polypeptidechain, called globin. In mammals, hemoglobin is present in erythrocytes,along with stroma which consists of proteins, phospholipids andcholesterol. See CLINICAL HEMATOLOGY, By Wintrobe, 6 Ed. Pages 138-199,(1967).

The reversible binding of oxygen requires the interaction between fourchains of hemoglobin (tetrameric hemaglobin) which results from theability of the protein to exist as two different quarternary structures(relaxed and tense) that have different oxygen affinities (Perutz, M. F., Prog. Clin. Biol. Res. 1: 3 (1975)). The two different oxygenaffinities permit hemoglobin to on-load oxygen when 1) the oxygentension is high (approximately 100 mm Hg pO₂) and to off-load oxygenwhen the oxygen tension is low (approximately 40 mm Hg pO₂) and giverise to a characteristic sigmoidal shape to the oxygen-hemoglobindissociation curve. It is now known that the tense state of somehemoglobin in red cells is stabilized by the presence of organicphosphates such as 2,3-diphosphoglycerate (2,3-DPG), with the tensestate of hemoglobin in solution not stabilized due to the absence of2,3-DPG. Accordingly, hemoglobin in solution has a lower P₅₀ thanhemoglobin in its natural form (Arnone, A., Nature 237: 146 (1972).

Aqueous hemoglobin exists in equilibrium between the tetrameric (MW68,000) and dimeric (MW 34,000) forms (Bunn, H. F. et al., Trans. Assn.Am. Physicians 81:187 (1968)). The dimers are excreted by the kidney andresult in rapid intravascular elimination of hemoglobin solutions, withsuch solutions having a 2-4 hour plasma half-life. Accordingly, effortshave been directed to overcome the inherent limitations of hemoglobinsolutions by molecular modification of the hemoglobin. The purpose ofthe molecular modification is to stabilize hemoglobin to prevent dimerformation and to maintain the tense conformational state. Bunn et al.,supra, demonstrated that cross-linking hemoglobin reduced renalelimination and increased intravascular retention time. Bunn et al.utilized bis (N-maleimidomethyl) ether; however, the resultinghemoglobin solution had a high oxygen affinity, i.e., a P₅₀ of 3 mm Hg.Pyridoxal-5-phosphate has been demonstrated to have an analogous effectto 2,3-DPG in lowering oxygen affinity, resulting in a P₅₀ of 26-30 mmHg (Benesch, R. E., Biochem. 11: 2568 (1972)). However, unlike 2,3-DPG,pyridoxal phosphate does not act as a cross-linking agent, resulting inintravascular retention times similar to that of unmodified hemoglobin(Greenburg, A. G. et al., Surgery 86: 13 (1979)). Thus it was thoughtthat pyridoxylation and cross-linking would be required to produce ablood substitute having low oxygen affinities (P₅₀ equal to 20-30 mm Hg)and adequate intravascular retention times (half disappearance times of20 or more hours).

In 1985, the Congress of the United States, Office of TechnologyAssessment (OTA), issued a report entitled "Blood Policy andTechnology." At chapter 6 of this report, alternative sources of bloodproducts were discussed, with the conclusion that the impetus to developalternative blood sources and substitutes based on economic, safety, andavailability considerations was a necessity. According to the report,the ideal red blood cell substitute would have six properties: 1) anoxygen dissociation curve and oxygen-carrying capacity similar to thatof intact red blood cells; 2) be non-toxic and non-antigenic; 3) havegood flow characteristics; 4) remain in the circulation for a longperiod of time; 5) have a long shelf life; and 6) be cost effective incomparison to present red blood cell transfusions. The report alsoconcluded that no substitute yet developed fulfills all these criteria.

Four basic approaches have been utilized to develop an adequate bloodsubstitute. In one approach, a class of synthetic compounds calledperfluoro chemicals are being developed. In a second approach,synthesized analogues of hemoglobin are being developed. Investigatorsare also attempting to assemble a red cell by encapsulating hemoglobinin lipid vesicles called liposomes. Finally, purified hemoglobin hasbeen chemically modified to prolong its circulation and enhance itsoxygen binding-dissociation properties.

According to the OTA report, supra, to date, none of these approacheshas proven satisfactory. The fluorocarbons are removed by thecirculatory system as foreign substances, and they become lodged in theliver, spleen, and other tissues. Artificial cells made of membraneencapsulated hemoglobin have not been used for many reasons. The use ofmicrocapsules made from synthetic polymers such as polystyrene,ethylcellulose, and silicone rubber introduces biologically incompatablematerials into a living system. The cell walls of the capsules tend toleak, it is difficult to control permeability of the wall, and thesecapsules are too rigid and too large to pass through the capillary bed.

The use of blood and blood fractions is fraught with disadvantages. Forexample, the use of whole blood often is accompanied by the risk oftransmission of hepatitis-producing virus and AIDS-producing virus whichcomplicate the patient's recovery in the first instance and is fatal inthe second. Additionally, the use of whole blood requires blood-typingand cross-matching to avoid immunohematological problems and interdonorincompatibility.

The blood fraction plasma (BFP) which is a physiologically balancedcolloidal solution that fulfills many of the requirements of a bloodvolume expander, cannot be safely used for this purpose. The highincidence and the risk of transmitting homologous serum hepatitisassociated with plasma is so great, that its use is no longer warranted.

The blood component hemoglobin possesses osmotic activity and theability to transport and exchange oxygen, but it has the disadvantage ofrapid elimination from circulation by the renal route and throughvascular walls, resulting in a very short, and therefore, unsatisfactoryhalf-life.

The literature, both patent and non-patent, is replete with efforts toproduce a satisfactory blood substitute from polymerized, cross-linked,stromal free hemoglobin. Bonsen et al., U.S. Pat. No. 4,001,200, andBonsen et al., U.S. Pat. No. 4,001,401 disclose polymerized,cross-linked, "stormal-free" hemoglobin and pharmaceutical compositions(and methods for using same) comprising the polymerized, cross-linked,"stromal-free" hemoglobin. The process for producing the polymerized,cross-linked, "stromal-free" hemoglobin of Bonsen et al. compriseslysing red blood cells, filtering through diatomaceous earth to removestroma, dialyzing to remove residual low molecular weight salts andmetabolytes, polymerizing to form water soluble, cross-linked,macromolecular, stromal-free hemoglobin, with a final sterilization byfiltering through a filter having a pore size of about 0.20 to 0.45microns. Included among the cross-linking agents disclosed by Bonsen etal. are dialdehydes such as glyoxal, malonic dialdehyde, succinicdialdehyde, glutaraldehyde, adipaldehyde, 3-methyl glutaraldehyde,propyladipaldehyde, phthalic dialdehyde, terephthaldehyde and malonicdialdehyde.

Bonsen et al. (III, U.S. Pat. No. 4,053,590), extends the disclosure ofBonsen et al. ('200) and Bonsen et al. ('401) with a discussion ofphysiologically acceptable polymeric plasma substitutes as carriers forthe blood substitute. Further, applications for use as an artificialoxygen exchange solution in conventional oxygenators such as cardiacby-pass, extracorporeal circulatory assist devices, and hollow-fiber andsheet type membrane devices for use in assisting the circulation in illpatients, is suggested. Additionally, the polyhemoglobin is suggested asa source of protein and oxygen in the microbiological assay of foods foraerobic bacillus and staphyllococcus to ensure the food is safe foranimal and human consumption and as a storing and preserving solutionfor viable isolated perfused mammalian organs for their eventualtransplant into a recipient.

Bonhard et al., U.S. Pat. No. 4,136,093 discloses a hemoglobinpreparation suitable for intravenous injection comprising asubstantially pyrogen-free condensation product of hemoglobin andpyridoxal phosphate. The hemoglobin preparation is claimed to have aretention time in the blood system of from 2 to 9 hours. The product isproduced by washing red blood cells with a weakly alkaline solution,hemolyzing, and treating the resulting material with a cation exchangeresin. The material is separated from the resin, diluted to a hemoglobinconcentration of about 5-9%, adjusted to a pH of about 7 to 9, treatedwith pyridoxal-5-phosphate and, optionally, treated with a solution of aborohydride and then a dialdehyde to cross-link the hemoglobinmolecules. The non-pyrogenic nature of the infusion solution is obtainedby, as a minimu, repeated washings with the weakly alkaline solution.

In Bonhard et al., U.S. Pat. No. 4,336,248, hemoglobin molecules werecoupled to increase their intravascular residence time withoutsignificantly diminishing the oxygen transport ability of the molecule.The hemoglobin molecules are coupled to one another and/or to serumproteins and gelatin derivatives using dialdehydes such as aliphaticdialdehydes of 3-8 carbon atoms. Optionally, pyridoxal phosphate may beadded subsequently. The coupled hemoglobin molecules are recovered byammonium sulphate precipitation.

In Simmonds et al., U.S. Pat. No. 4,401,652, there is disclosed aprocess for preparing a "stromal-free" hemoglobin solution. The Simmondset al. process is particularly adapted for large scale production of"stromal-free" hemoglobin, with reduced methemoglobin formation. Theprocess comprises washing blood cells to remove non-cellular components,removing leukocytes, typically by filtration through a suitableadsorbent which preferentially retains the leukocytes, lysing theremaining red blood cells ultrasonically or mechanically, precipitationof the hemoglobin by mixture with a polyvalent cation, a polysulphate,and a polyvalent anion, and final purification by filtration anddialysis. The resulting hemoglobin solution is "substantially pure" and"free of stroma" and other lipoprotein cellular constituents andcontains less than 5% methemoglobin.

Tye, U.S. Pat. No. 4,529,719, discloses "stromal-free" tetramerichemoglobin which is cross-linked with certain bis-disalicyl esters andmodified with pyridoxyl-5'-phosphate followed by reduction to producebis-diamide covalently cross-linked, pyridoxyl-5'-phosphate covalentlymodified, tetrameric hemoglobin. The modified cross-linked"stromal-free" tetrameric hemoglobin is disclosed to be disease-free andcapable of transporting oxygen to perfused tissue and remains in theintravascular space. Additionally, the product is suggested to be freefrom cell surface antigens, making it suitable for transfusion in placeof red blood cells.

The modified cross-linked, "stromal-free" tetrameric hemoglobin of Tyeis produced by starting with red blood cells of freshly drawn, outdated,or frozen packed cells or whole blood. The blood is drawn in sterilefashion into containers with sufficient anticoagulant activity toprevent clot formation. Hemoglobin from a variety of mammalian sources,such as human, bovine, ovine, or porcine are disclosed to be useful. Anynon-heme protein is removed, preferably by zinc precipitation.Hemoglobin is released from the red blood cells by hypotonic lysisfollowed by ultrafiltration. The filtered hemoglobin is passed through asubsequent filtration step to remove virus particles, proteinaggregates, and stromal elements. The typical filter has a nominal poresize of 0.020 microns and an exclusion for globular proteins of1,000,000 Daltons. Zinc iron is added to precipitate the hemoglobin andthe precipitate concentrated by filtration. The non-heme protein isremoved in the filtrate. The resulting hemoglobin is then cross-linkedusing the bis-disalicyl esters and treated with pyridoxyl-5'-phosphate,followed by reduction of the reversible Schiff base covalent bond.

Kothe et al., U.S. Pat. No. 4,526,715 discloses a method for producinghighly purified hemoglobin solutions free of plasma proteins andresidual stromal lipids prepared from human blood or from animal bloodin quantities large enough for clinical applications. The disclosedprocess comprises contacting red blood cells with a washing solution,hemolysing by introduction of the concentrated red blood cells into 2-3times the volume of water, separating the stroma from the hemoglobin byultrafiltration, and concentration in a third filtration stage utilizinga second ultrafiltration unit having a permeability of 10,000 to 50,000Daltons.

However, in spite of the recent advances in the preparation of"stromal-free," cross-linked hemoglobin origin blood substitutes, theneed has continued to exist for a blood substitute which issubstantially free of endotoxins, phospholipids, and non-hemoglobinproteins, which is capable of 1) transporting adequate amounts of oxygento tissue under ambient conditions; 2) having an oncotic activityequivalent to that of whole blood; 3) having an adequate intravascularretention time; 4) transfusible to all recipients without cross-matchingor sensitivity testing; 5) free from disease agents such as bacteria andvirus particles (hepatitis, AIDS, etc.); and 6) storable with minimumamounts of refrigeration.

SUMMARY OF THE INVENTION

Recognizing the long-standing need in the field to develop a bloodsubstitute comprising an oncotically active protein solution capable oftransporting oxygen and readily available when massive transfusions arerequired, the inventors endeavored to develop a blood substitute basedon a hemoglobin solution. Further, recognizing that the massive demandsfor such a blood substitute would require volumes of starting materialfar in excess of that which could potentially be made available asdiscarded human blood, a further goal of the present invention was togenerate a process for creating such a blood substitute wherein nonhumanmammalian blood sources would be suitable as starting materials.

With these goals in mind, the following invention has resulted, asemi-synthetic blood substitute comprising monomeric mammalianhemoglobin in cross-linked form, said semi-synthetic blood substitutebeing substantially free of endotoxins, phospholipids and non-hemoglobinproteins such as enzymes. An additional aspect of the present inventioncomprises the process by which the afore-mentioned blood substitute isprepared. Essentially, the blood substitute is prepared from a mammalianblood fraction by a process comprising 1) separation of red blood cellsfrom the mammalian blood fraction; 2) hemolysis of the red blood cellsto produce a composite of monomeric hemoglobin and stroma, includingphospholipids; 3) separation by filtration of the hemoglobin,contaminated with at least a portion of the phospholipid; 4)purification of the monomeric hemoglobin by high performance liquidchromatography (HPLC) to separate the hemoglobin from all other proteinsresidual of the red blood cells, as well as the phospholipid, enzyme andendotoxin contaminants; 5) cross-linking (polymerizing or aggregating)the monomeric hemoglobin; and 6) partially separating the cross-linkedhemoglobin from the non-cross-linked hemoglobin. An essential aspect ofthe present process comprises conducting the above steps underconditions which result in a product which is substantially free ofendotoxins, phospholipids and non-hemoglobin proteins such as enzymes,and has a defined molecular weight distribution of greater than about90% between 68,000 daltons and 500,000 daltons.

The resulting product (hereinafter "Invention Hemoglobin") is a bloodsubstitute which is substantially free of endotoxins, has vascularpersistence of at least two days, has the property of reversibly bindinggaseous ligands such as oxygen and is useful for transporting andsupplying oxygen to vital tissues and organs. As such, the bloodsubstitute of the present invention is useful as a blood expander andresuscitating fluid in the management of disease and for maintainingcirculatory integrity where needed, i.e., in response to sudden andmassive blood loss.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H are flow sheets describing the process of Example I. FIG. 1Arefers to the blood collection phase. FIG. 1B refers to the bloodseparation phase. FIG. 1C refers to the microporous filtration phase.FIG. 1D refers to the ultrafiltration phase. FIG. 1E refers to thecolumn chromatography phase. FIG. 1F and 1G refer to the crosslinkingphase. FIG. 1H refers to the storage and process fluid preparationphase.

FIG. 2 is a graphic comparison of platelet numbers between three groupsof rabbits, with percent change from baseline represented on theordinate and time represented on the abscissa. T₁ represents thebaseline platelet level; T₂ represents the platelet level at 15 minutespost-transfusion; T₃ represents the platelet level at 1 hourpost-transfusion; T₄ represents platelet level at 3 hourspost-transfusion; and T₅ represents platelet level at 24 hourspost-transfusion. The solid circles represent the mean value±standarderror for platelet levels for six rabbits having one-third of estimatedblood volume replaced with a hemoglobin solution containing between oneand two endotoxin units (EU)/ml; the clear circles represent the meanvalue±standard error for platelet levels of four rabbits that hadone-third of blood volume replaced with 5% Plasma Protein Fraction(commercial product found to contain 0.05-0.15 EU/ml); and the squaresrepresent the mean value±standard error for six rabbits that hadone-third of the estimated blood volume replaced with pure polymerizedhemoglobin according to the present invention, said hemoglobincontaining 0-0.35 EU/ml.

FIG. 3 is a graphic representation comparing white blood cell levels forone-third transfused rabbits from baseline to 24 hours. As in FIG. 2,the ordinate represents percent change, with the abscissa representingtime. T₁ represents the baseline platelet level; T₂ represents theplatelet level at 15 minutes post-transfusion; T₃ represents theplatelet level at 1 hour post-transfusion; T₄ represents platelet levelat 3 hours post-transfusion; and T₅ represents platelet level at 24hours post-transfusion. The solid circles represent the meanvalue±standard error of 6 rabbits that had one-third of the estimatedblood volume replaced with a hemoglobin solution containing between 1and 2 EU/ml; the clear circles represent the mean value±standard errorof four rabbits that had one-third of the estimated blood volumereplaced with 5% Plasma Protein Fraction (commercial product found tocontain 0.05-0.15 EU/ml); and the squares represent the meanvalue±standard error of 6 rabbits that had one-third of the blood volumereplaced with pure polymerized hemoglobin according to the presentinvention, containing 0-0.35 EU/ml.

FIG. 4 is a graphic comparison of serum fibrinogen levels between threegroups of rabbits. The ordinate represents the percent change infibrinogen levels; the abscissa represents the time period, with T₁representing baseline; T₂ the value at 15 minutes post-transfusion; T₃the value at one hour post-transfusion; T₄ the value at 3 hourspost-transfusion and T₅ the value at 24 hours post-transfusion. Thesolid circles represent the mean value±standard error for six rabbitswith one-third of the estimated blood volume replaced with a hemoglobinsolution that contained between 1 and 2 EU/ml; the clear circlesrepresent the mean value±standard error for four rabbits that hadone-third of blood volume replaced with 5% Plasma Protein Fraction(commercial product found to contain 0.05-0.15 EU/ml); and the squaresrepresent the mean value±standard error of six rabbits that hadone-third of blood volume replaced with pure polymerized hemoglobin,containing 0-0.35 EU/ml.

FIG. 5 represents a graphic comparison of prothrombin levels betweenthree groups of rabbits. The ordinate represents the percent change inprothrombin levels, with the abscissa representing time. T₁ representsthe baseline prothrombin level; T₂ represents prothrombin levels at 15minutes post-transfusion; T₃ represents prothrombin levels at 1 hourpost-transfusion; T₄ represents prothrombin levels at 3 hourspost-transfusion; and T₅ represents prothrombin levels at 24 hourspost-transfusion. Solid circles represent the mean value±standard errorof six rabbits that had one-third of the estimated blood volume replacedwith a hemoglobin containing between 1 and 2 EU/ml; the clear circlesrepresent the mean value±standard error of four rabbits that hadone-third of the estimated blood volume replaced with 5% Plasma ProteinFraction (commercial product found to contain 0.05-0.15 EU/ml); and thesquares represent the mean value±standard error of six rabbits that hadone-third of the estimated blood volume replaced with pure polymerizedhemoglobin, containing 0-0.35 EU/ml.

FIG. 6 is a graphic representation comparing serum creatinine levelsbetween three groups of rabbits. The ordinate represents the percentchange in serum creatinine levels, with the abscissa representing time.T₁ represents the baseline serum creatinine level; T₂ represents serumcreatinine levels at 15 minutes post-transfusion; T₃ represents serumcreatinine levels at 1 hour post-transfusion; T₄ represents serumcreatinine levels at 3 hours post-transfusion; and T₅ represents serumcreatinine levels at 24 hours post-transfusion. Solid circles representthe mean value±standard error of six rabbits that had one-third of theestimated blood volume replaced with a hemoglobin solution containingbetween 1 and 2 EU/ml (Chromogenic LAL test); the clear circlesrepresent the mean value±standard error of four rabbits that hadone-third of the estimated blood volume replaced with 5% Plasma ProteinFraction (commercial product found to contain 0.05-0.15 EU/ml); and thesquares represent the mean value±standard error of six rabbits that hadone-third of the estimated blood volume replaced with pure polymerizedhemoglobin, containing 0-0.35 EU/ml.

FIG. 7 represents a graphic comparison of the changes in hematocrit(Hct) following a 50% hemorrhage-transfusion in monkeys. The ordinaterepresents the hematocrit with the abscissa representing the time indays. Hematocrits of hemoglobin-transfused monkeys are noted with thesolid circles; hematocrits of Plasma Protein Fraction are denoted withthe open circles.

FIG. 8 is a graphic representation of white blood cell percent, redblood cell percent, hemoglobin percent, and hematocrit percent, fromtime 0 through day 21, of a dog which underwent a 40% exchangetransfusion with a cross-linked hemoglobin solution of the presentinvention.

FIG. 9 is a graphic representation of white blood cell percent, redblood cell percent, hemoglobin percent, and hematocrit percent, fromtime 0 through day 18, of a dog which underwent a 25% exchangetransfusion with a cross-linked hemoglobin solution according to thepresent invention.

FIG. 10 is a graphic representation of white blood cell percent, redblood cell percent, hemoglobin percent, and hematocrit percent, fromtime 0 through day 78, of a dog which underwent a 33% exchangetransfusion with a cross-linked hemoglobin solution according to thepresent invention.

FIG. 11 is a graphic representation of white blood cell percent, redblood cell percent, hemoglobin percent, and hematocrit percent, fromtime 0 through day 47, of a dog which underwent a 33% transfusion with5% human albumin solution.

FIG. 12 is a graphic representation of white blood cell percent, redblood cell percent, hemoglobin percent, and hematocrit percent, fromtime 0 through day 23, of a dog which underwent a 50% blood volumehemorrhage, followed by immediate replacement with 5% albumin solutionas a first step in the exchange transfusion. This was followed by arapid removal of another 50% of the blood volume followed by replacementwith a cross-linked hemoglobin solution of the present invention.

FIG. 13 is a graphic representation of white blood cell percent, redblood cell percent, hemoglobin percent, and hematocrit percent, fromtime 0 through day 30, of a dog which underwent a 50% hemorrhagefollowed by immediate replacement with an equal volume of a cross-linkedhemoglobin solution of the present invention and a second 50% hemorrhagefollowed by replacement with an equal volume composed of a cross-linkedhemoglobin solution of the present invention and a 5% albumin solution,mixed in equal parts.

FIG. 14 is a graphic representation of Table V of Example VIII. Theordinate represents the percentage of total Invention Hemoglobinremaining in each one of its compositional subgroup molecular weights.The abscissa represents time in hours.

FIG. 15 is a graphic representation of the data collected from theexperimental work of Example IX. The graph demonstrates that as redblood cells are progressively exchanged for Invention Hemoglobin,beginning at or below a hematocrit of 20%, there is an expected increasein the total plasma hemoglobin concentration.

FIG. 16 is a graphic representation of data collected from theexperimental work of Example IX. Seven test animals and six controlanimals were exchanged from initial hematocrit levels with non-oxygenbearing volume replacements (i.e., Ringer's lactate and hydroxyethylstarch solutions) down to approximately 20%. Both groups showed asimilar decrease from initial values to approximately 20%. Below 20%,the control group showed a progressive decrease in arterial oxygencontent associated with progressive decrease in hematocrit. The sixcontrol animals did not survive. In contrast, the test group displayednoticeably higher arterial oxygen content which was maintained despitethe progressive decrease in hematocrit. The seven test animals survivedand appeared clinically normal. In FIG. 16, the ordinate representsarterial oxygen content while the abscissa represents hematocrit.

FIG. 17 is a graphic representation of experimental data collected fromExample IX. The ordinate represents mixed venous oxygen content, whilethe abscissa represents hematocrit.

FIG. 18 is a graphic representation of data collected from Example IX.The ordinate represents contribution to oxygen content; the abscissarepresents hematocrit. The graph demonstrates the contribution ofInvention Hemoglobin and of sheep red blood cells to arterial oxygencontent at various hematocrit levels. In the seven test animals, asInvention Hemoglobin increased and hematocrit decreased, an increase inpercentage of arterial oxygen content was contributed by the InventionHemoglobin. At hematocrit levels in the range of 3%, almost 90% ofarterial oxygen content is contributed by the Invention Hemoglobin, withthe remainder being contributed by the residual sheep red blood cellsand diluted plasma.

FIG. 19 is a graphic representation of oxygen delivery during exchangetransfusion. The ordinate represents the percent of initial value; theabscissa represents time in hours. During the first exchange withRinger's lactate solution, hematocrit as well as oxygen deliverydecreased. At this point (→), exchanges were begun with InventionHemoglobin solution. While sheep hematocrit decreased further, oxygendelivery increased back towards baseline levels, in association with theInvention Hemoglobin solution infusion. This demonstrates that at theend of the exchange, oxygen delivery is due principally to the InventionHemoglobin and not to the residual hematocrit (approximately 3%).

FIG. 20A-B is a graphic representation of data collected from Example X.In both FIGS. 20(A) and 20(B), the ordinate represents hematocritpercent to blood volume while the abscissa represents time in minutes.Both the test and control groups showed a similar decrease in hematocritduring the exchange transfusion.

FIG. 21A-B is a graphic representation of experimental data collectedduring the course of Example X. FIG. 21(A) is data collected from fourtest dogs, while FIG. 21(B) is data collected from three control dogstransfused with hydroxyethyl starch solution (HES). The test group showsa progressive increase in plasma (free) hemoglobin to approximately 6percent during exchange, in contrast to the control group. The ordinaterepresents the grams per 100 ml. of blood volume.

FIG. 22 is a graphic representation of data collected from Example X.Comparison of four test dogs (upper figure) and three control dogs(lower figure) demonstrates that the test animals receiving InventionHemoglobin maintained stable cardiac outputs in contrast to the controlgroup which showed increasing cardiac outputs associated with declininghematocrit and arterial oxygen content. In FIG. 22, the ordinaterepresents the liters per minute of cardiac output (flow) while theabscissa represents time in minutes.

FIG. 23A-B represents a graphic representation of data collected duringthe performance of Example X. A comparison of four test dogs (upperfigure) and three control dogs (lower figure) shows reduced but adequateand well-maintained arterial oxygen content during the exchange in thetest group in contrast to the control group, which had progressivedecline in oxygen content associated with the decreasing hematocrit. InFIG. 23, the ordinate represents arterial oxygen content while theabscissa represents time in minutes.

FIG. 24A-B represents a graphic comparison of four test dogs (upperfigure) and three control dogs (lower figure). The ordinate in FIG. 23represents venous oxygen content while the abscissa represents time inminutes. The test group shows reduced but adequate and well-maintainedvenous oxygen contents during the exchange in contrast to the controlgroup which had progressive decline in oxygen content associated withthe decreasing hematocrit.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The product of the present invention comprises a mammalian bloodsubstitute which is a cross-linked hemoglobin solution having amolecular weight distribution of greater than about 90% in the range of68,000 to 500,000 Daltons, osmolarity as measured by freezing pointdepression in the range of 180-320 milliosmoles per liter of solution, afinal hemoglobin content of 5-25, preferably 9-13 grams per deciliter, amethemoglobin content of less than about 20% and preferably less thanabout 10%, physiologic levels of sodium chloride and potassium chloride,less than about 1 nanomole of phospholipid per milliliter, less thanabout 1 part per million of cross-linking agent, a P₅₀ in the range ofabout 18-36, preferably about 24-32 mm Hg, and an intravascularhalf-life of at least 4 days, with at least a portion of the materialremaining in the body for at least 6 to 8 days.

The term "P₅₀ " is recognized in the art to describe the interactionbetween oxygen and hemoglobin and represents the partial pressure ofoxygen (pO₂) at a 50% saturation of hemoglobin. This interaction isfrequently represented as an oxygen dissociation curve with the percentsaturation of hemoglobin plotted on the ordinate axis and the partialpressure of oxygen in millimeters of mercury (mm Hg) or torrs plotted onthe abcissa.

By the term "intravascular half-life" is intended the period of time inwhich the initial amount of hemoglobin in an in vivo environment fallsto half its initial value.

The blood substitute is further characterized by a cross-linking profileon gel permeation chromatography of 50-70% cross-linking, with nomaterial having a molecular weight of less than 68,000 being detectable.

The profile for gel permeation chromatography of the blood substitutecan be characterized by integration from low molecular weight to totalexcluded volume where the amount of cross-linking is from 50% to 75 or80%. A preferred embodiment of the invention shows a molecular weightdistribution of greater than about 90% in the range from 68,000 MW to500,000 MW where no more than 10 to 15% of the material is in theexcluded volume which is in the range of 400,000 to 500,000 MW andhigher. After careful filtration, the gel permeation chromatogram alsoshows that almost none of the material, if any, is below the 68,000 MWlevel. The initial 68,000 molecular weight peak of pure hemoglobin asmeasured by gel permeation chromatography is broadened afterpolymerization such that the retention time of 68,000 MW is somewhatcomplexed so that it is larger--up to 90,000 MW. Integration can beperformed on this final peak such that it is found that at least 20%will be in the 68,000 MW range. This fraction does not cause a toxicresponse in the animal, but is merely excreted by the kidneys and can beshown in the urine upon sampling.

Additionally, the blood substitute is substantially endotoxin free andpyrogen free as well, and does not cause any of the following abnormaland detrimental chemical and physiologic functions in vivo: (1) does notactivate complement; (2) does not cause hemorrhagic disorders; (3) doesnot cause abnormal platelet function or aggregation; (4) does not causeabnormal prothrombin times (PT); (5) does not cause abnormal partialthromboplastin times; (6) does not interfere with blood typing orcross-matching; (7) is non-toxic to the kidneys in 3.5 grams perkilogram per body weight or 8 grams per deciliter circulating bloodvolume; (8) exhibits circulating persistance of at least seven days; and(9) acts as a stimulus to accelerated erythropoiesis.

By the term "blood substitute" is intended to be a material having theability to transport and supply oxygen to vital organs and tissues andto maintain intravascular oncotic pressure. Accordingly, the termencompasses materials known in the art as "plasma expanders" and"resuscitation fluids" as well.

The term "cross-linked" or "polymerized" is intended to encompass bothinter-molecular and intramolecular polyhemoglobin, with at least 50% ofthe polyhemoglobin of greater than tetrameric form.

The term "substantially endotoxin free", for the purposes of the presentinvention, may be described functionally as a blood substitute whichcontains less than 1.0 endotoxin units per milliliter of solution, at aconcentration of 10 grams of hemoglobin per deciliter of solution. Thisblood substitute, when used as a replacement for approximately one-thirdof the total blood volume of a rabbit, produces a percent change inblood platelet levels, over time, which is substantially similar toCurve delta-delta of FIG. 2, or a percent change in white blood celllevels, over time, which is substantially similar to Curve delta-deltaof FIG. 3, or a percent change in fibrinogen levels, over time, which issubstantially similar to Curve delta-delta of FIG. 4, or a percentchange in pro-thrombin levels, over time, which is similar to Curvedelta-delta of FIG. 5, or a percent change in serum creatinine levels,over time, which is substantially similar to Curve delta-delta of FIG.6.

In a preferred embodiment, the "substantially endotoxin free" bloodsubstitute of the invention will contain less than 0.5, and preferablyless than 0.25, most preferably less than 0.02 endotoxin units permilliliter of solution (EU/ml) as measured by the Limulus AmebocyticLysate (LAL) assay. The LAL assay is described by Nachum et al.,Laboratory Medicine, 13:112-117 (1982) and Pearson III et al.,Bioscience, 30:461-464 (1980), incorporated by reference herein.

By the term "endotoxin(s)" is intended the generally cell-boundlipopolysaccharides produced as a part of the outer layer of bacterialcell walls, which under many conditions are toxic. When injected into ananimal, endotoxins cause fever, diarrhea, hemorrhagic shock, and othertissue damage.

By the term "endotoxin unit" (EU) is intended that meaning given by theUnited States Pharmacopeial Convention of 1983, Page 3014, which definedEU as the activity contained in 0.2 nanograms of the U.S. referencestandard lot EC-2. One vial of EC-2 contains, 5,000 EU.

The present invention further involves the process for producing thesemi-synthetic, substantially stromal-free blood substitute. The processcomprises the steps of (1) obtaining the blood raw product, (2)fractionating the blood raw product to produce a red blood cell fractionwhich is substantially free from white blood cells and platelets, (3)mechanically disrupting the red blood cell fraction to produce ahemoglobin-containing solution, (4) clarifying the hemoglobin-containingsolution to produce a hemoglobin solution which is substantially free ofcellular debris, (5) microporously filtering the hemoglobin solutionwhich is substantially free of cellular debris to produce a partiallysterilized hemoglobin-containing solution, (6) ultrafiltering thepartially sterilized hemoglobin-containing solution to produce asize-separated hemoglobin-containing solution, (7) chromatographicallyseparating the size-separated hemoglobin-containing solution to producea hemoglobin substantially free of phospholipids and non-hemoglobinproteins, said hemoglobin retained on the chromatographic column, (8)eluting the substantially phospholipid-free hemoglobin from the columnto produce a substantially endotoxin-free hemoglobin solution, (9)cross-linking said substantially endotoxin-free hemoglobin solution toproduce the cross-linked blood substitute, and (10) partially separatingthe cross-linked blood substitute by filtration, all steps done in asubstantially endotoxin-free environment.

Each of the process steps will be described in greater detail below.

I. THE PROCESS A. Blood Collection

Starting point in the present invention is an erythrocyte (red bloodcell) source. As such, the starting material may be freshly drawn humanblood, outdated old blood from blood banks, placentas, or packederythrocytes obtained from human donor centers. Additionally,erythrocytes obtained from animal blood are entirely suitable as well.Accordingly, blood from a variety of sources such as bovine, ovine, orporcine may be used. Because of its ready availability, bovine bloodobtained from slaughterhouses is the preferred erythrocyte source.

The unique approach of the present invention has required specialtechniques in the collection and handling of blood in large quantities.Large collection trochars are used which extract the blood in a sterilemanner. The trochars require careful insertion and handling and areconnected to tubing approximately 2 feet in length. In order to insertthe trochar the hide must be cut away, peeled back, and the trochar theninserted in the animal's major vessels close to the heart with care notto puncture the esophagus. Avoiding the introduction of bacteria and themaintainance of endotoxin-free or low endotoxin level material isimportant. This is accomplished using individual containers that arepre-charged with an anticoagulant and that are depyrogenated andre-checked for endotoxins. Typical anticoagulants include sodiumcitrate. In all cases, endotoxin levels of the containers must be lessthan 0.01 endotoxin units as detected by LAL.

This solution is then charged to small vessels that can hold between 2to 10 gallons of gathered blood in a sterile manner and, therefore,maintain the blood in an endotoxin-free state. The collected blood inits container is capped off immediately to avoid exposure to theenvironment. Upon completion of the collection process, the material ischilled, typically to about 4° C., to limit bacterial growth. There isno pooling of blood at this time; the blood is later checked forendotoxins and sterility to ensure that (1) no one cow is sick; or (2) abad collection technique has not contaminated the entire batch orcollection for that day. Although the above collection method ispreferred, there are many collection methods which are suitable andavailable to one with ordinary skill in the art.

B. Red Cell Separation

The blood is brought to a processing center at which time each vessel issampled and checked by LAL analysis for endotoxin levels. If theendotoxin level is higher than 6-7 EU per ml the blood is discarded.Only if the individual blood container tests at below that endotoxinlevel is the material approved for secondary processing.

Typical secondary processing of the prior art was to suspend the blood(ACD anticoagulated blood) in a saline solution of physiologic saltconcentration and centrifuge in order to separate effectively the plasmaproteins and white cells from the red cells. This suspension process isperformed through several "washing" steps, i.e., 2-4 times, in anattempt to remove all free proteins. In the process of the presentinvention, however, it was found that this approach was untenable forpractical manufacturing scale up; in fact, to separate the hemoglobinproduct free from many contaminants, it is not necessary to do thiswashing process at all.

In the preferred process, the whole blood from the animal, once it hasbeen checked for endotoxins, is passed through a semi-continuous typecentrifuge where the red cells, white cells and plasma can beeffectively separated on as large a scale as desired. The processemploys a bowl-type semi-continuous centrifuge where the bowl ismaintained at 15,000 to 18,000 rpms, i.e., a Sharples AS-15 unit. Thebowl and the top configuration are set up with an opening at aparticular radius that permits a discrete layer separation such that redcells, white cells and plasma can be removed through the operation.While the Sharples bowl-type centrifuge is preferred, typical separationapparatus also suitable include basket centrifuges such as aremanufactured by Beckman Instruments.

To prepare for this operation the centrifuge is depyrogenated, i.e.using, typically, a 0.5 molar sodium hydroxide for at least 1 hour priorto installation into the machine housing container. The top spouts orcollection devices are handled in a similar fashion, thus allowing forcomplete depyrogenation of all contact surfaces the blood may encounter.Once the parts have been put through the depyrogenating process, thesystem is assembled. A lobe pump or peristaltic pump with sanitarydesign is used to flush fluid through the entire system and collectionports; typically, a solution of 0.5 molar sodium hydroxide is used, butother depyrogenating solutions known to the art are suitable as well. Atthe completion of the flush, it is necessary to reduce the pH to a rangewhich is conducive to the handling of the hemoglobin solution. This isaccomplished through a water purge which reduces the pH to a range ofapproximately 7-9. In some cases it has been necessary, because ofdensity differences between the depyrogenating solution and water, touse an acid solution to help neutralize the strong base employed in thedepyrogenation step. All these solutions must be depyrogenated andchecked prior to use in the washes.

Once the pH level has been brought below 9, samples are obtained fromthe effluent streams of the centrifuge and endotoxin testing isperformed. When an endotoxin level of 0.01 EU/ml or less is achieved,the system is ready for the separation of the blood. However, in thisseparation it is critical that the flow rate of the blood into thecentrifuge be at a sufficient rate to limit the amout of sedimentationof red blood cells caused in the Sharples centrifuge. If the flow rateis too low, the red cells will settle into the bowl of the centrifugeand not be separated or collectible into a separate container. In acentrifuge with a 4" diameter bowl, a flow rate in excess of 2.5 to 3liters per minute, but not exceeding 6 liters per minute, is required tolimit sedimentation. If a larger bowl or different g forces areemployed, then different flow rates are required, the particularparameters being within the skill of the art.

Once all parameters have been established, the blood from the variousbatches or various cows is introduced into the system and the effluent(separated red cells) is collected under sterile conditions in aseparate container. However, at this point the effluent is pooled and isno longer treated on an individual animal basis. To eliminate anyvariations of pH or potential initial entrapment of bacteria on thecentrifuge, positive pressure sterile nitrogen is applied to the chamberwhere the bowl is spinning. For true sterilization of the system, asteam sterilization cycle may be applied by introducing steam into thebowl spinning chamber and steaming for up to an hour prior to use. Aftercompletion of steaming, the system is cooled i.e., through glycolcoolant tubes, typically to about 4° C. (After harvesting from theanimal, it is important that the blood be brought to and maintained at atemperature just above freezing, typically about 4° C.)

In the separation of the red cells it is important that the collectionchamber, i.e., spout area, where the red cells are collected at the topof the high speed spinning bowl, be configured such that high impact ofthe cells occurs. In striking these surfaces, the red cells becomebroken through a mechanical degradation, as opposed to using a hypotonicsolution. (In a hypotonic solution, the red cells swell and cause themembrane to rupture from hydraulic forces.) This is a change from thenormal operation of swelling cells to lyse them by hydraulic pressure,to one of mechanical degradation. This mechanical degradation isextremely rapid and does not generate the high degree of free small cellmembrane components found by other methods. The red cells are collectedin a vessel and prepared for the second centrifuge operation.

C. Red Cell Clarification

Once the blood has been processed and the red cells have been separatedfrom the white cells and plasma, the mechanically disrupted red cellsare diluted using pure depyrogenated water which has been maintained atlow temperature, i.e., about 4° C. Typically, disrupted the red cellsare diluted by at least 50%. The red cells are then introduced into thesecond separation step; typically a similar type of centrifuge to thefirst operation may be used. In a preferred embodiment, a Sharplescentrifuge with a 4" bowl operating in a semi-continuous mode andspinning at 15,000 to 18,000 rpms is used. The flow rate, however, issubstantially decreased: 0.5 liters per minute or less is recommended.Unlike the first processing step, this step employs a different type oftop configuration to the centrifugation bowl. No separation of twolayers such as the plasma white cell and red cell composition iseffected at this step. This clarification step results in the separationof all cell debris from the liberated hemoglobin solution. The same caretaken in the first step for depyrogenation and sterility must beemployed in the second step. Once this material has been collected fromthis second step, it is ready for microporous filtration.

D. Microporous Filtration

The microporous filtration must be operated differently than a pressurefiltration mode. In a practical sense, pressure filtration is notacceptable to industrial scale processing of hemoglobin solutions. Toemploy microporous filtration successfully, either a plate and framefiltration or hollow fiber filtration system may be used; however, itmust be operated such that the pressure drop across the membrane (thetransmembrane pressure) is carefully maintained to within about 5 poundsper square inch (psi). If the pressure drop exceeds the tolerance levelby 1 to 2 psi, the membrane rapidly becomes plugged with the remainingcell debris and the flux rate across the membrane drops to anunacceptable level for industrial purification in a semi-continuousmode.

While tangential flow of this material across the membrane is at a flowrate of 2 to 5 liters per minute, the flux through the membrane is onthe order of 0.1 to 0.2 liters per minute. This operational rate ismaintained to eliminate cellular debris from building up on themembrane. When the concentration of solution tangential to the membranedecreases to less than 10% of the initial solution, the remainingsolution is discarded or it is re-diluted with water to extractadditional product and thereby produce a higher yield of hemoglobin fromthe system.

The filtration system may utilize lobe or peristaltic pumps withsanitary design, thus decreasing and limiting seals and shafts that maycause introduction of bacteria and pyrogen contamination. Other pumpdesigns known to the art for sanitary pumping may be used, however. Suchpumps include centrifugal, gear, and tubular diaphragm pumps.

The membrane systems are pre-treated to ensure depyrogenation and properpH. If handled improperly, pyrogens are added at this point and itbecomes more and more difficult to remove them throughout the remainingprocessing steps. Depyrogenation and pH control is accomplished by usingstandard sanitation procedures and depyrogenation procedures, i.e.,typically with sodium hydroxide and voluminous washings withpyrogen-free water to bring the pH to within acceptable ranges forhandling of the hemoglobin solution (<pH 9). While handling of atransmembrane pressure limitation in such a manner is not well known andhas only been practiced in the last few years on a selected basis withtissue fluid processing, suitable techniques are within the skill of theart.

In a preferred embodiment, a filtrate side restriction is employed suchthat flux rate is limited to its steady state (non-plugging) condition.If a fluid stream is applied to a tangential flow membrane system, andthe inlet pressure is approximately 20 psig, the outlet pressure is 0psig, and the filtrate side of the membrane is 0 psig, giving an averagetransmembrane pressure (ATP) of 10 psi., the solution to be filtered hasa tendency to fill and extrude into the porous membrane surface. It willessentially plug the membrane and will not be swept clear by thetangential shear created by the cross-flow of fluid. By restricting theoutlet (filtrate) so that the ATP is only 1 to 2 psi, the tangentialflow sweeps the surface clear and flux across the membrane remainsconstant yet low when compared to initial flux rates with high ATP. Theflow under steady state conditions may be 0.2 liters per minute, with 1to 2 psi ATP, and 1 to 1.2 liters per minute with 20 psi ATP. However,the 20 psi ATP will stay constant and cause the flux to rapidly fall tozero flux within minutes.

With the completion of this first microporous filtration step, thesolution has been at least partially sterilized and substantially allcell debris above 0.45 micron has been removed. In some cases it may berequired to make the solution sterile at this point. In these cases,after the 0.45 micron microporous filtration has been completed a 0.22micron filtration may be employed in the same manner as the 0.45 micronfiltration. The resulting solution is now ready for the molecularseparations which follow.

E. Ultrafiltration

The next step includes the careful staging of 100,000 molecular weightfiltration (measured in Daltons) using membranes which effectivelyretain everything greater than 100,000 molecular weight and which permiteverything less than 100,000 molecular weight to pass through. Typicalmembranes are commercially available from Millipore Corporation, and aresold under the trade name Durapore. Everything below these levels isfiltered through the membrane system. Hemoglobin (about 67-68,000molecular weight) passes through this membrane system and is collectedin tankage.

This large membrane filtration operation requires careful monitoringbecause, over a period of hours, the membrane will become plugged andfiltration flux will decrease rapidly. It is, therefore, necessary toflush the membrane on a regular basis with a pure water solution. Theflushing reduces cell debris which may otherwise coat and occlude themembrane, thereby reducing hemoglobin solution flux rate. The tangentialcross-flow time cycling over this membrane can be up to 2 hours and doesnot affect the methemoglobin level or the viability of the hemoglobinfor its intended purpose. When the fluid volume after the microporousfiltration has been reduced to about 30% of its volume duringultrafiltration, sterile pyrogen free water may be added to obtaingreater yield of hemoglobin solution. Maximum dilution is about 50%.This material may also be discarded. If the 30% original material isdiluted, it again may be reduced to about 30%, at which time it isdiscarded. The filtered intermediate is held in sterile, pyrogen-freetankage for subsequent operations. A typical device for effecting theultrafiltration step is a Millipore Pellicon cassette with a Duraporemembrane; however, other devices known to the art may be used as well.

The next ultrafiltration step, requires a removal of material below68,000 molecular weight. This isolates small molecule hemoglobin andother small proteins that may have been carried over from the wholeblood plasma. In all cases the hemoglobin solution is maintained at aconcentration of about 5 to 15 grams per deciliter. Filtrationaccomplished at this step provides some degree of concentration. At highconcentration, low flux rates are exhibited. In both ultrafiltrationoperations where 100,000 mw and 30,000 mw membranes are employed, thenecessary depyrogenation steps and subsequent checking after washingwith pyrogen-free water are usually required.

In the 100,000 mw separation step pyrogens may be removed since somepyrogens are between 100,000 and 1 million mw. With the depyrogenationof the 30,000 molecular weight membrane and the preparation of thismembrane packet for its filtration process, the hemoglobin solution haspassed over this tangential flow system to allow the perfusion of smallmolecules through the membrane. Recycle may or may not be used in thisoperation, although it is required in the 100,000 mw filtration step.The retentate (material retained), is held in a storage tank and checkedfor endotoxins. In all cases the endotoxins must be below 0.5 EU per mlbecause subsequent operations makes removal of high levels of pyrogensquite difficult. This material is stored under a sterile nitrogen orargon atmosphere that maintains stability in the tankage system.Typically the methemoglobin level is below 1% at this point in theprocess. The filtration steps must be performed at low temperatures,typically at about 4° C. Following the filtration, the material iseither frozen or directly aliquoted to lot sizes for large scalechromatographic processing.

F. Chromatography

Prior to the chromatographic separation, the material is in aconcentrated state of not less than 2 grams per deciliter and notgreater than 11 grams per deciliter. The chromatographic system includespumps, a gradient generator, columns and detectors.

A typical pumping system comprises a diaphragm pumping system with arange of 1 to 5 liters per minute pumping capability. Such a systemincludes a Pulsafeeder 8480 stainless steel diaphragm pump orequivalent. For the feed system a smaller pump is used where the flowwill range from 0.1 liters per minute to 1.5 liters per minute. Thispump is typically a smaller volume pump and would be of a tubulardiaphragm design. A typical pump for this operation is a Pulsafeeder7120. To configure the chromatographic system such that it operatesproperly it is required that two large systems are assembled so that onewould be used as the operating system for chromatographing the materialwhile the other system is used for flushing, cleaning and regenerationof the column.

A solvent composition generation system has been fabricated and iscomprised of flow control valves that deliver to the applicable pumpingsystem a proportional amount of two fluids generating a fluidcomposition gradient over time of a specific ionic strength. Ionicinteraction is used to effect an ion exchange chromatographic separationon the column system. Fabrication of this or equivalent systems iswithin the skill of the art.

A typical flow control valve is a Baumann flow control valve, which hasbeen programmed to operate using a standard programmable controller, forexample a Texas Instrument 530 programmable controller. All piping andtubing to the system is of sanitary nature and made of 316L tubingapproximately 1/2" to 1" in diameter. The feed system through thegradient generator and through the pump are presented to a separationsegment or column as is known in the art.

The column is typically made from stainless pipe. The stainless pipe canbe interconnected with tubing of 1/2" diameter, such that it willcomprise one long column for effecting a separation. The pipe or columnis typically lined with teflon to give compliance to the internalsurface which is helpful in effecting packing of the media internal tothe column system.

The chromatographic system effluent can be monitored by a splitting ofthe stream and passing that small representative amount through arefractive index detector like a model R401 from Waters Associates or anultraviolet detector, typically a 441 Waters Associates model number.These systems can be used to monitor the effluent stream from the columnto detect the point at which the protein of interest is being eluted.

Once all parameters have been established and guidelines have been set,there is no need for a detector in the system and fraction collectionmay be achieved through simple time elution profiles.

These materials may be either fabricated or purchased from varioussuppliers of industrial grade piping and tubing. The column isfabricated to achieve uniform distribution of sample being presented tothe top of the column and, in conjunction with that, uniform samplecollection from the effluent of the column. The length to diameter ratiois significant in that creating a column that is too long or too shortwill significantly affect the efficiency of the separation andequilibration for doing the ion exchange.

The column is comprised of separation media which allows for someirreversible adsorption of phospholipids (irreversible in the simpleoperation mode) and a discrete ion exchange separation using a specifiedgradient elution pattern of solvent. The separation media compriseparticles of silica gel of from about 50 to 150 microns in size; theflow across this material is in the range of about 2.5 liters perminute.

The silica gel is of an average pore size of 300 angstrom units, asmeasured by BET nitrogen absorption. This silica gel is available fromvarious manufacturers i.e., W. R. Grace Davison Chemical Co. This gel isthe preferred substrate on which to build the derivatized surface whichgives the functionalized property for separation of the hemoglobinsolution.

To produce the separation media, it is necessary to derivatize thesilica surface first with a special silane which creates a diol chemicaltype surface on the silica surface. This diol can be typically achievedby creating a glycidoxypropyltrimethoxysilane coating to the surface,with techniques which are well known in the field of chromatography,typically by suspending the silica and the silane in a vessel which hasbeen partially diluted with water. The reaction is a water base reactionand this polymer will coat onto the surface of the silica. This reactionto coat the silica requires a 20 hour reaction time at approx. 70° C.Once this coating has been achieved on the silica, the material may besimply washed by a series of methanol and acetone washes to create aclean permanently bonded, diol coated silica. The material is then driedand prepared for the second step or series of steps where differentmonomers are coated onto the surface and the surface will be derivatizedto have a quaternary amine type surface property for doing or preformingthe specific kind of ion exchange separation. The organic stationaryphase is a thin skin of cross-linked polymer. The cross-linked polymerthat is put onto the surface is built up from two different hydrophilicvinyl monomers. For example, one may use a monomer such asn-methyl/acrylamide in 48% water solution (Silar Labs), andmethylamidopropyltrimethylammonium/chloride.

The two monomers have various capabilities; one monomer willcopolymerize with another functional monomer, i.e., one having the ionexchange or absorption properties desired. It will cross-link with otherpolymer chains and anchor the cross-linked polymer to the silicasurface.

The specific monomers chosen for this purpose have a vinyl functionalityand a reactive groups that react in such a way that they can react witheach other, forming the bridges that are necessary to coat to thesurface and the coating of a stationary phase consisting of an aminefunctional group, thereby producing an ion exchange capability in thedesired range.

Once these two monomers have been suspended in the aqueous solution, aswell as with a methanol solution of the silica, the suspension solutionis evaporated away leaving the monomers coated onto and into the silicagel. At this stage the mixture is resuspended in a new solution whichalso includes a radical initiation system, such as a Dupont product,Vazo 64. To initiate the reaction, the reaction mixture is heated to thepoint where it must be maintained at 70°-75° C., no higher and no lower.

At this temperature the reaction proceeds and the polymer is coated ontoand bonded to surface, including the functional groups that produce thesurface property used in the chromatographic media. When the reactionhas been completed, it is necessary to remove unreacted monomer with aseries of washes with several solvents, such as acetone and methanol.Following the completion of all these washes the material is dried andready for use.

A typical column diameter is 6" and a typical column length is 2 feet.However, suitable variations are within the skill of the art. Themaximum operating pressure is 500 psi. The injection is made by pumpingthe solution onto the column, typically at a rate of 1 liter per minutefor approximately 1 minute, then injection is terminated. Therefore, theload factor is no greater than 1 liter of material at 7 grams perdeciliter. At the completion of loading the hemoglobin solution, anisocratic flow of buffer (e.g. tris buffer at pH 8.9 to 9.0) is appliedto the column and continues to flow through the column until such timeas the gradient or variable composition flow is started. The buffer, asthe primary eluant, is then diluted over time. Typically, the eluant ismade up from a Tris buffer base solution which is made in aconcentration of 1.8 grams per liter Tris with a pH of about 8.6-9.2.The temperature range for elution is about 3°-10° C. These ranges aresignificant since changing the temperature range also changes the pH ofthe elution solution. The secondary solution for eluting the material ofinterest may be prepared using a solution of Tris buffer, highlypurified in the same manner as the previous buffer. In addition, thisbuffer also contains salt to a 1 molar concentration. This solution isalso pH adjusted to be identical to the original pH solution, which isin the range of 8.6 to 9.2. Release of phospholipids takes place priorto the elution of the hemoglobin, with endotoxins eluting after thehemoglobin peak of interest has been collected.

The chromatographic selection technique is done by UV absorption,refractive index, typically using equipment as described above, orvisible observation of the effluent stream. Typically, the first portionof the eluting hemoglobin is discarded to waste; then the collection ofeffluent begins and continues until the peak or the response has beenreduced to 20% to 10% of its peak amplitude. This constitutes thefraction to be collected and the fraction of interest for purification.If the collection point runs beyond the appropriate retention time, thenother proteins and/or endotoxins may be collected and the product may berendered unusable. Similarly, if collected before a peak retention time,the material may contain unacceptable levels of endotoxins. Thephospholipid count and extraneous sub-components of hemoglobin arediscarded, both the pre-retention peaks and the post-retained peaks.This collection process allows intermediate product material which hasbeen diluted approximately 40 to 1, in a pH range of 8.9 to 9.0.

In this pH range it is necessary to concentrate the material rapidly. Inthis dilute state, the appearance and formation of methemoglobin occursat a rapid rate. To effect this concentration, a membrane of 10,000 mwor less can be used. Either plate and frame tangential flow orhollow-fiber flow systems are acceptable. Typical systems include aMillipore Pellicon cassette. When concentration levels of 7 to 10 gramsper deciliter are achieved and a methemoglobin level of less than 1.5%is achieved, the fractions are collected for long-term storage. At thispoint the material may also be transported into a reactor system for thesubsequent polymerization reaction.

Following the collection in the chromatographic system, thechromatographic column undergoes a sequence of washes to prepare it fora second loading of unpurified material. If this column preparation isnot performed, various subcomponents and contaminants will elute andrender subsequent runs invalid. Typically, the wash is accomplished byusing a 100% pyrogen-free 0.5-1.0 molar NaCl wash for a period of atleast 5 minutes, or 3 column volumes, and no more than 10 minutes, or 6column volumes. At the completion of the buffer gradient and salt flush,the fluid phase is returned to initial conditions of 100% tris bufferwhich is 0.18 grams per liter of tris buffer, and pH is adjusted toapproximately 8.9±.1 for the hemoglobin elution process. Although rangesof the hemoglobin pH have been studied, the 8.9 -9 pH range of thechromatographic system yields the highest and best isolation of a purehemoglobin analogue. At lower ranges (8.6-8.4), hemoglobin is eluted ina pure state but the loadability of material onto the separationmaterial is drastically decreased. At pH levels of 9.5-11, the formationof methemoglobin occurs at a rate which makes it untenable to maintainlow methemoglobin levels. Further, there is the potential forcross-contamination. Over a period of 2 hours the methemoglobin levelmay increase 5% at this higher pH range. The material eluting from thecolumn is a hemoglobin solution which is substantially free of otherproteins, endotoxins, and phospholipids. This material has utility inits own right as an intermediate product in the production of across-linked , substantially endotoxin free, substantiallyphospholipid-free semi-synthetic blood substitute.

Long term storage of hemoglobin solution after concentration with sodiumchloride and tris buffer has been carried out for periods of as short as1 day and as long as 6 months. The results have shown no productdegradation or increase in the methemoglobin level if the product ismaintained at -20° C. The solution, however, upon thawing over a 2 to 24hour period, may exhibit an increase in methemoglobin. If left in anunfrozen state, the methemoglobin level will continue to rise. In otherstudies where material had low pH (pH 7 and below) the methemoglobinlevel increase is dramatic, i.e., a 10 percentage point increase within3 hours.

The hemoglobin solution typically has the following characteristics:

    ______________________________________                                        HEMOGLOBIN SOLUTION SPECIFICATIONS                                            ______________________________________                                        Hemoglobin g/dl    7-15                                                       Oxyhemoglobin      90-100%                                                    Carboxyhemoglobin  0-2%                                                       Methemoglobin      0-10%                                                      pH                 6.5-9.0                                                    Endotoxin EU/ml    <0.01                                                      Molecular weight daltons                                                                         68,000                                                     Phospholipids      <1 nanomole/ml                                             TLC Plat iodine                                                               developed -- clear                                                            Amino Acid Analysis                                                                              No foreign protein                                                            amino acids                                                N-Terminal sequencing                                                                            98%+ conforms to Bovine                                                       hemoglobin sequence                                        Page Gel           Single Band? (no virus                                                        contamination)                                             Salt concentrations may vary                                                  High performance chromatography                                                                  99.9%+ hemoglobin protein                                  ______________________________________                                    

G. Polymerization (Cross-linking) Reaction

When material has been either specifically allocated for polymerizationreaction or has been thawed from the frozen state, it is introduced intoa sterile pyrogen-free reactor having impellar blades positioned toeffect rapid mixing and high shear. (A typical apparatus is a 3 literApplicon fermenter with a flat bladed impeller positioned one inch fromthe bottom of the reactor and with 5 one-half inch baffles positionedabout the reactor. This is necessary to prevent large polymer formationwhen the cross-linking agent is added.) The hemoglobin solution added tothe reactor is put on a recirculation system and the hemoglobin solutionis withdrawn from the reactor and passed across an exclusion membrane,typically a 10,000 mw exclusion filter, and returned to the reactor in alow O₂ environment. (The reactor may be blanketed with an inert gas,i.e. argon.) This last procedure is accomplished by drawing a vacuum onthe reactor and placing an argon blanket on the liquid in this reactor.Extreme caution is taken to eliminate introduction of bacteria at thispoint; the material is pyrogen-free, exhibiting no endotoxins by LALanalysis.

A sterile pyrogen-free buffer (pH 8.9-9.1) is then added to the reactorthrough a depyrogenating membrane filter, typically a 10,000 moleculeweight filter. Simultaneously a 10,000 mw concentration loop is cycledto balance the volume of introduced fluid and the exiting fluid from thereactor system.

The reaction buffer which is being used to neutralize the high pH is aphysiologic composition of sodium, chloride, and potassium, with typicalvalues of 120 milliequivalents sodium, 120 milliequivalents chloride and4 milliequivalents potassium. The pH of the solution is adjusted withHCl and tris base to a pH of about 4.7 to 5.2. If the pH is too lowduring the pH reducing process, large amounts of methemoglobin form atthe point of introduction of the neutralizing acid solution. Thefiltration process is maintained until the pH has dropped to a range ofabout 7.4 to 8.0 pH units in the reactor. At this time the introductionis terminated and the introduction of the cross-linking solution ismade.

Suitable cross-linking agents are disclosed in U.S. Pat. No. 4,001,200to Bonsen et al., incorporated by reference herein. The preferred classof cross-linking agents are those having aldehyde functionality, mostpreferably, dialdehydes, with glutaraldehye being the cross-linkingagent of choice.

Where glutaraldehyde is used, the glutaraldehyde is added, typically ata rate of about 100 milliliters per hour. The glutaraldehyde solution istypically prepared by thawing a high purity specification glutaraldehyde(stored at -20° C. to 4° C.) in a short, typically 2-5 minute, timeframe. This solution, which preferably has about a 25% concentration ofglutaraldehyde, is then added to pyrogen-free water, the proportions ofwhich make up a solution which is preferably about 5 milliliters of a25% solution diluted into 100 milliliters of pyrogen-free water. Thesolution is added at the rate specified above to the reactor andreaction mixture.

The monitoring of cross-linking solution and its effects oncross-linking (polymerization) is done by gel permeation chromatography.The gel permeation chromatography requires the use of a 300 Angstrompore size hydrophilic packing material column with resolution capabilityof over 24,000 plates per meter. A typical column is available fromWaters Associates; a typical packing material is Waters Protein Pak 300SW. The eluting chromatogram as recorded is integrated over the time ofpeak elution and quantitated against the starting material. Preferably,a cross-linking percentage of 50% to 70% is achieved. This number isdetermined by the percentage of material eluting from the column whichis less than 600,000 molecular weight (Daltons) and greater than 68,000molecular weight (Daltons).

H. Membrane Concentration

Once greater than 50-55% cross-linking, as calculated by gel permeationchromatography, has been achieved, the solution is ready for 100,000molecular weight membrane concentration. During this membrane filtrationthe tangential flow of the reaction mixture is passed over the membranewith a permeation of material which is 68,000 or less through themembrane system. This is performed until about a 25% reduction in fluidhas been achieved.

At the point when cross-linking is deemed to be complete, a quenchingsolution, i.e., a solution of pyrogen-free lysine, pH 7, is added. Theconcentration of the lysine solution is 1 gram per liter. This lysinesolution is added to quench the polymerization reaction ofglutaraldehyde with hemoglobin and to complex with excessglutaraldehyde. It is also believed that this material will fix tounpolymerized glutaraldehyde bound to hemoglobin molecules. At thecompletion of this addition, molecular weight distribution is determinedand found to have been stabilized as measured by gel permeationchromatography. Filtration is then started to remove excess lysine,excess glutaraldehyde and any other molecular weight species which isbelow 100,000 M.W.

The gel permeation chromatogram of the initial non-cross-linkedhemoglobin solution exhibits molecular weight sizes from 16,000 to68,000 Daltons, with the largest amount at 68,000 Daltons. Afterfiltration, there is some, not more than 50%, 68,000 Dalton hemoglobinand the appearance of material under 68,000 Dalton molecular weight isnot detectable. Filtration of the material after cross-linking alsoprovides opportunity to balance the electrolytes and pH of the solutionand thereby give a balanced physiologic solution for injection.

At the completion of this filtration process the material is removedfrom the system and bagged ready for freezing. At the completion of allprocesses and during the bagging, a sample of material is withdrawn fortesting.

II. THE PRODUCT

Typically, the product has the following characteristics. The molecularweight distribution of the material has greater than 90% of the materialin the range of 68,000 Daltons to 500,000 Daltons. Osmolarity asmeasured by freezing point depression is typically from 220 to 320milliosmoles per liter of solution. The electrophoretic patternexhibited on gel electrophoresis shows bands in 68,000 to 500,000molecular weight range. The final hemoglobin content can be adjusted to5 to 25, preferably 9 to 13 grams per deciliter and the methemoglobinlevel is under 20%, preferably under 10%. The ion concentrations ofsodium chloride and potassium are nontoxic to the animal or to thespecies to be tested. Thin layer chromatography developed for thedetection of phospholipids exhibit a clear plate upon developing byiodine staining. Phospholipids as determined by phosphoric acidreduction are non-detectable, with less than one nanomole per milliliteras the limit of detection. Gas chromatography is used as a quantitativemeasure for free glutaraldehyde. With detection of 1 part per million bygas chromatography as the limit, no glutaraldehyde may be detected. Noprotein other than hemoglobin is present as determined by gelchromatography and iso-electric focusing techniques.

The solution generally has less than 0.01 endotoxin units per ml asmeasured by LAL (limulus amoebocytic lysate) assay with a 0.01 to 0.1sensitivity scale, and is pyrogen-free by all testing. Rabbit studieshave been performed on this material which exhibits the samecharacteristics as would be exhibited by a pyrogen free material in thatno fever is exhibited by the rabbits. This material does not produce anyabnormal endotoxin response and other factors in the rabbits beingtested as it related to hemorrhagic conditions done on a control groupof rabbits which were then supplied with a pure plasma fraction of 1/3volume. It should be noted that in all cases, there was some elevatedlevels of enzymes and some histopathology that demonstrated changes inthe organs. Most of these changes however, were deemed to be reversibleand were, as mentioned earlier, similar to those found due tohemorrhagic conditions and replacement by a pure plasma proteinfraction. This can be translated to a lack of endotoxin response andother factors in higher animals. The purity as monitored by highperformance liquid chromatography using an ion exchange capacity forseparation exhibits four discrete peaks which upon quantitation areconsistent between batches regardless of the molecular weightdistribution characterized by gel permeation chromatography. Thesubstance produced exhibits life sustaining capabilities in oxygentransport as demonstrated by P₅₀ values of 20 to 28 mm of mercury.Further, importantly, the hemogloblin solutions of this inventiondemonstrate less clinically significant vaso-constrictive propertiesthan those demonstrated by other prior art cross-linked hemoglobinsolutions. The material further exhibits properties of increasedcellular appearance of red blood cells in various mammalian species, anddoes not cause any of the following abnormal and detrimental chemicaland physiologic functions in vivo: (1) does not activate complement; (2)does not cause hemorrhagic disorders; (3) does not cause abnormalplatelet function or aggregation; (4) does not cause abnormalprothromboplastin times (PT); (5) does not cause abnormal partialthromboplastin times; (6) does not interfere with blood typing orcross-matching; (7) is non-toxic to the kidneys in 3.5 grams perkilogram per body weight or 8 grams per deciliter circulating bloodvolume; (8) exhibits circulating persistence of at least seven days; and(9) acts as a stimulus to accelerated erythropoiesis. The material istypically as characterized in the Table below.

    ______________________________________                                        CHARACTERIZATION OF TYPICAL BLOOD                                             SUBSTITUTE OF THIS INVENTION                                                  ______________________________________                                        STERILITY    Sterile by standard culture technique                            NON-         <0.01 EU/ml sample when tested by LAL                            DETECTABLE   and compared against standard curve that                         ENDOTOXIN    ranges in sensitivity from 0.01 EU/ml to                         LEVEL        0.125 EU/ml.                                                     BAG CONTENTS Na 120 ± 20 milliequivalent Cl 115 ± 20                                 milliequivalent K 4.0 ± 1 milliequivalent                                  Hemoglobin 11 grams ± 2 per deciliter                                      Lysine < 1 gram/liter                                                         Glutaraldehyde -- none detectable Tris                                        <1.5 gram/liter                                                               Pyrogen Free H.sub.2 O volume -- 450-500 ml                                   Methemoglobin < 10%                                                           Phospholipid < 1 nanomole/ml                                                  Hemoglobin molecular weight distribution                                      % greater than 68,000 - at least 50%                                          % greater than 500,000 - 8% ± 2%                                           Osmolarity by freezing point depression                                       220-320 milliosmoles per liter of                                             solution                                                         CONTAINER    Fenwal Bag Code 4R2023 600 ml sterile,                                        nonpyrogenic fluid path                                                       Supplied by Fenwal Laboratories                                  STABILITY    -20° C. no change for greater than 8 months                            4° C. 5 days with methemoglobin level                                  under 10%                                                        ______________________________________                                    

III. UTILITY

The blood substitute of the present invention may be utilized in amanner similar to the suggested prior art blood substitutes and bloodexpanders. Thus the blood substitute may be used for replacing bloodlost by acute hemorrhage, to replace blood loss occurring duringsurgical operations, in resuscitation procedures after accidental bloodloss, for supplying oxygen, and generally to maintain blood volume inrelated conditions. As a plasma expander, the blood substitute may beutilized in volume deficiency shock, an alleviant in anaphylactic andallergic shock, for replacing plasma lost after burns, and as a resultof diarrhea.

The blood substitute of the present invention may be utilized as suchfor all mammalian species, but is particularly useful in the treatmentof humans. The blood substitute of the present invention is misciblewith recipient blood and its components, is substantially non-toxic,non-antigenic, non-pyrogenic, and, especially, substantially free ofendotoxins and other cell-bound and cell-free proteins. Itscolloid-oncotic properties make the product especially useful formaintaining the level of the blood and plasma in the management ofdisease states as well. Further, the material is extremely valuable inthat it may be used without an accompanying risk of transmission ofdisease. Further, it is believed that the blood substitute of thepresent invention is devoid of the immunologic problems that areassociated with the administration of whole blood, and does not causeany of the following abnormal and detrimental chemical and physiologicfunctions in vivo: (1) does not activate complement; (2) does not causehemorrhagic disorders; (3) does not cause abnormal platelet function oraggregation; (4) does not cause abnormal prothrombin times (PT); (5)does not cause abnormal partial thromboplastin times; (6) does notinterfere with blood typing or cross-matching; (7) is non-toxic to thekidneys in 3.5 grams per kilogram per body weight or 8 grams perdeciliter circulating blood volume; (8) exhibits circulating persistenceof at least seven days; and (9) acts as a stimulus to acceleratederythropoiesis. The blood substitute of the present invention may beadministered using techniques of administration which are conventionalin the art, as disclosed in Blood Transfusion, by Hustis.

As a blood expander, the blood substitute of the present invention maybe mixed with water-soluble physiologically acceptable polymeric plasmasubstitute such as polyethylene oxide, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, and ethylene oxide-propylene glycolcondensate. The material may also be mixed with colloidal plasma-likesubstitutes and blood plasma expanders such as linear polysaccharides,including dextrans having a molecular weight of 40,000 to 70,000, gumarabic pectins, balanced fluid gelatin, and hydroxyethyl starch.

Additionally, the blood substitute of the present invention maybe usedas an artifical oxygen exchange solution in conventional oxygenators.When used for assisting the circulation in ill patients, oxygenators arewidely used to mechanically oxygenate venous blood extracorporeally,utilizing one or more pumps for maintaining circulation and forperfusion of oxygen by the exchange of gases between blood in anisolated vascular bed and oxygen across an oxygenation membrane.

EXAMPLES EXAMPLE I The Blood Production Process

In the following Example, the equipment identified in parenthesis isfurther identified in the reference list which follows. FIGS. 1A-1H area flow sheet for the process of this Example, with the equipmentreference numbers of the Example corresponding to the Figure referencenumbers.

A. Blood Collection

Erythrocytes (red blood cells) were obtained from the blood ofslaughtered cows. Because of its ready availability, the bovine bloodobtained from slaughter houses is the preferred erythrocyte source.

Collection trochars were used to extract the blood in a sterile manner;the animal hide is cut, peeled back and the trochars then inserted inthe animal's major vessels close to the heart. The introduction ofbacteria was avoided and the maintenance of pyrogen-free or low-pyrogenlevel material was accomplished using a container, specifically BloodCollection Tank (T-102), which was precharged with depyrogenated sodiumcitrate as anticoagulant (0.5 liters). A suitable Blood Collection Tankis a container with a volume of 25 liters, wherein the container isvented to atmosphere through sterile vent filter (F).

The depyrogenated sodium citrate anticoagulant was prepared by addingsodium citrate, through Citrate Line (100), and Water-For-Injection(WFI), through Water Line (101), to Citrate Mix Tank (T-101), whereinthe sodium citrate anticoagulant was mixed by Citrate Mixer (M-101).Citrate Pump (P-101) then took a suction on Citrate Mixer (M-101)through Citrate Pump Inlet (102) and discharged sodium citrateanticoagulant through Discharge Time (103) and Citrate Filter (F-101). A10 KD filter is acceptable to depyrogenate the sodium citrateanticoagulant. The depyrogenated sodium citrate anticoagulant was thendischarged from Citrate Filter (F-101) through Citrate Addition Line(104) and into Blood Collection Tank (T-102).

Following the addition of sodium citrate, Blood Collection Tank (T-102)was filled with blood through Blood Line (105). The collected blood inBlood Collection Tank (T-102) was capped off immediately to avoidexposure to the environment. Upon completion of the collection process,the material was chilled to about 4° C. to limit bacterial growth. Therewas no pooling of blood from different cows at this time. The blood waschecked for pyrogens and sterility to ensure that (1) no one cow wassick or (2) that no contamination occurred during collection. The bloodwas transported in a refrigerated condition from the slaughter house tothe process plant.

B. Red Cell Separation

Blood was pumped using Feed Pump (P-301) from Blood Collection Tank(T-102) through in-series Blood Addition Line (300) and Feed Pump Inlet(303) and was discharged through Feed Discharge Line (304) to SeparationCentrifuge (CT-301). An acceptable flow rate for Feed Pump (P-301) isbetween 2.5 and 6 liters per minute. A suitable separation centrifuge isa Sharples AS-15 unit.

Prior to receiving blood, Separation Centrifuge (CT-301) was sanitizedby depyrogenation through flushing Separation Centrifuge (CT-301) withsodium hydroxide solution. An example of a suitable flush would be for aperiod of at least one hour with 0.5 molar sodium hydroxide. NaOH CIPTank (T-303) received NaOH through Caustic Line (322) and WFI throughWater Line (321). A pump, not shown, then flushed Separation Centrifuge(CT-301) with sodium hydroxide solution by taking a suction on NaOH CIPTank (T-303) through Caustic Addition Line (301) and discharged thesodium hydroxide solution into Separation Centrifuge (CT-301) throughCombined Injection Pipe (302), Feed Pump Inlet (303), Feed Pump (P-301)and Feed Discharge Line (304). Then to reduce the pH to less than 9, WFIfrom WFI CIP Tank (T-304), which was previously filled through WaterLine (323), was injected into Separation Centrifuge (CT-301).

An alternate method for sterilizing Separation Centrifuge (CT-301) wasby steaming. After steaming, the centrifuge was cooled to about 4° C. byglycol coolant flowing through Cooling Line (312).

While being fed blood, Separation Centrifuge (CT-301) was operatedbetween 15,000 and 18,000 rpm to separate red blood cells from whiteblood cells and plasma. The separated white cells and plasma were thendischarged through Discharge Line (313). The separated red cells werethen mechanically degraded in Separation Centrifuge (CT-301) due to thehigh impact of the red cells on mechanical surfaces. The effluent fromthe centrifuge (separated, degraded red cells) discharged through RedCell Discharge Line (305) into a 114 liter Dilution Tank (T-301) whereinthe degraded red cells collected under sterile conditions. At this pointthe effluent was pooled and was no longer treated on an individualanimal basis. Also during centrifuge operation, sterile nitrogen wasapplied at positive pressure to the centrifuge through Nitrogen Line(311) to exclude bacteria.

An acceptable dilution tank includes Sterile Vent Filter (F) andNitrogen Supply Line (315).

C. Red Cell Clarification

The degraded red cells were diluted in Dilution Tank (T-301) by adding,through Water Line (314), Water-For-Injection (WFI) which had beenmaintained at 4° C. The red cells were then pumped by Clarification FeedPump (P-302) at less than 0.5 liters per minute from Dilution Tank(T-301) through Pump Inlet (306) and then discharged through DischargeLine (307) and into Clarification Feed Cooler (E-301) before beinginjected through Feed Line (308) into Clarification Centrifuge (CT-302).Clarification Feed Cooler (E-301) maintains the degraded red blood cellflow at a temperature of about 4° C. by using a glycol coolant.

While operating between 15,000 and 18,000 rpm, Clarification Centrifuge(CT-302) is blanketed with nitrogen through Nitrogen Line (316) and isalso maintained at a suitable temperature by glycol coolant flow throughCooling Line (318). This clarification step resulted in the separationof all cell debris, which are then discharged through Solids Line (317),from the liberated hemoglobin solution which flowed by gravity, throughHemoglobin Discharge Line (309), to Sterile Holding Tank (T-302). Asuitable sterile holding tank includes Sterile Vent Filter (F) andNitrogen Supply (320).

D. Microporous Filtration

Stage I Microfilter Feed Pump (P-401) took a suction through Pump Inlet(310) on the hemoglobin solution from the Sterile Holding Tank (T-302)and discharged through Discharge Line (400) into Stage I Microfilter(F-401) (0.45 micron). An acceptable flow rate for Microfilter Feed Pump(P-401) is between 2-5 liters per minute with the flow rate adjusted tomaintain an acceptable average transmembrane pressure across Stage IMicrofilter (F-401). The retenate from the filter was recycled, throughRecirc Line (401), to Sterile Holding Tank (T-302) while hemoglobinpassed through the membrane of Stage I Microfilter (F-401) as filtrate.WFI was then added to the retenate in Sterile Holding Tank (T-302)through Water Line (319) to allow additional filtration and extractionof hemoglobin from the retentate. The filtrate was pumped at 0.5 litersper minute by the Stage I Microfiltrate Pump (P-402), through Pump Inlet(402) and Discharge Line (403), and then into the Stage I MicrofiltrateTank (T-402). Tank T-402 used in this microporous filtration stage was aglass vessel of 100 liter capacity and is equipped with WFI Line (415)and a Caustic flushing connection, not shown, with Sterile Vent Filter(F) and Nitrogen Supply Line (409). The microporous filters are of plateand frame construction (such as a Millipore Pellicon Cassette type) andwere operated such that the average transmembrane pressure (ATP) wascarefully maintained to between 1 and 2 pounds per square inch. Whilethe tangential flow of the material across the membrane was 2 to 5liters per minute, the flux through the membrane was on the order of 0.1to 0.2 liters per minute. This operational rate was maintained toeliminate cellular debris from building up on the membrane. When theconcentration of the solution tangential to the membrane decreased toless than 10% of the initial solution, the remaining solution wasdiscarded. (Alternatively, it is re-diluted in Sterile Holding Tank(T-302) with Water-For-Injection, through Water Line (319), to achieve ahigh yield of hemoglobin from the system.)

Subsequent to Stage I microfiltration, Stage II Microfilter Feed Pump(P-403) took a suction through Pump Inlet (404) on the hemoglobin inStage I Microfiltrate Tank (T-402) and discharged the hemoglobin throughDischarge Line (405) into Stage II Microfilter (F-402) (0.22 microns).The retentate from the filter was recycled through Recirc Line (410)back to Stage I Microfiltrate Tank (T-402), while the hemoglobin passedthrough the filter membrane as filtrate. WFI was then added to theretenate in Stage I Microfiltrate Tank (T-402) through Water Line (415)to allow additional filtration and extraction of hemoglobin from theretentate.

Filtrate was pumped by Stage II Microfiltrate Pump (P-404) through PumpInlet (406) and Discharge Line (407) and then into Stage IIMicrofiltrate Tank (T-401). Tank (T-401) is equipped with WFI Line(411), and with Caustic Flushing Line (412), which join in a with CommonInjection Pipe (CIP), and also with Sterile Vent Filter (F), NitrogenSupply Line (413), Waste Discharge Line (414) and Recirculation Line(416). The solution was now ready for the molecular separations whichfollowed.

E. Ultrafiltration

Hemoglobin was pumped from the Stage II Microfiltrate Tank (T-401)through Pump Inlet (408) and Discharge Lines (500, 501) and then into100,000 molecular weight Stage I Ultrafilters (F-501A and B) using theStage I Ultrafilter Feed Pump (P-501). The retentate from the filterswas recycled from Recirc Lines (512, 513, 514, 416) and into Stage IIMicrofiltrate Tank (T-401). The filtrate flowed through Filtrate Lines(502, 503, 504) to the Stage I Ultrafiltrate Tank (T-501). The flow rateon the retentate side was 5 liters per minute. The filtrate flow ratewas 0.2 liters per minute. Hemoglobin was then pumped from the Stage IUltrafiltrate Tank (T-501) through Pump Inlet (505) and Discharge Lines(506, 507, 508), using the Stage II Ultrafilter Feed Pump (P-502), andthen, through the Stage II (30,000 D) Ultrafilters (F-502A and B). Theretentate either recycled to the Stage I Ultrafiltrate Tank (T-501)through Recirc Line (522) or flowed through Filtrate Line (510) to theStage II Ultrafilter Tank (T-502). The filtrate was sent to wastethrough Waste Line (518). The tanks (T-501, T-502) were glass vessels of100 liter capacity with sterile vent filters (F) and are provided withconnections for washing with WFI, specifically Water Lines (515, 519),and with Caustic, specifically NaOH Lines (516, 520), wherein the WFIand Caustic Lines join to form common injection pipes (CIP), and forsupplying nitrogen, specifically Nitrogen Lines (517, 521). Connectionswere provided beneath the Stage II Ultrafiltrate Tank (T-502) forflushing the downstream system with WFI and Caustic, not shown.

F. Chromatography

The chromatography stage was automated based on a TI-530 softwarepackage. Key variables were protected by a lock ensuring repeatabilityof the process operations. Hemoglobin was pumped by Column Feed Pump(P-601) from the Stage II Ultrafiltrate Tank (T-502) through Tank Outlet(511) and Pump Inlet (608) and is then discharged through DischargeHeader (600), and onto Column (C-601A), through Column A Injection Lines(612, 613, 614, 615, 616) and Air Operated Valves (AOV 1, AOV 5, AOV 7,AOV 10, AOV 12) or separately onto Column (C-601B) through Column BInjection Lines (625, 626, 627, 628, 629) and Air Operated Valves (AOV1, AOV 6, AOV 9, AOV 11, AOV 13). An appropriate injection onto a Column(C-601A, C-601B) is injection for one minute with a flow rate for ColumnFeed Pump (P-601) of approximately 1 liter per minute. WFI can also bepumped by Column Feed Pump (P-601) from Water Line (607) to eithercolumn (C-601A, C-601B).

Air Operated Valve (AOV 8) operates to isolate Column (C-601A) fromColumn (C-601B) and also provides the flow path for injection intoColumns (C-601A, C-601B) during the column wash cycle.

After injection of hemoglobin the gradient or variable composition flowwas started and injected onto the column from using Gradient Pump(P-602) which took a suction on Mixing Chamber (M-601) through PumpInlet Header (643). Gradient Pump (P-602) discharged through DischargeLine (644) and Column Selector Air Operated Valve (AOV 2) and then,either injected gradient into the Column (C-601A) through GradientInjection Line (645) and Column A Injection Lines (613, 614, 615, 616)and Air Operated Valves (AOV 5, AOV 7, AOV 10, AOV 12), or injectedgradient into the Column (C-601B) through Gradient Injection Line (646)and Column B Injection Lines (626, 627, 628, 629) and Air OperatedValves (AOV 6, AOV 9, AOV 11, AOV 13).

The composition of the gradient flow was established utilizingcomputer-controlled Proportioning Valves (AOV 15, AOV 16) whichcontrolled the rate of flow of each reagent from Reagent Lines (1020,1023), through Reagent 1 Supply Line (609) and Reagent 2 Supply Line(610), through Reagent Supply Header 642, and into Mixing Chamber(M-601) wherein the reagents thoroughly mixed to form the gradient.

Reagent 1 was produced, in Reagent Tank No. 1 (T-1003), from mixing TRISand NaCl, added through Reagent Line (1024), and WFI, added throughWater Line (1025). Reagent 1 was then filtered by using Reagent No. 1Pump (P-1003) to pump the Reagent 1 in Tank (T-1003) through Pump Inlet(1018) and Discharge Line (1019) into Reagent No. 1 Filter (F-1003) andback to the tank (T-1003) through Filtrate Line 1030. The pressurecontrol valve (PCV) in Retentate Line (1035) controls the amount ofreagent flow that is allowed to flow past the filter (F-1003) and returndirectly to the tank (T-1003). Reagent Tank No. 1 (T-1003) is a 150gallon tank which includes Sterile Vent Filter (F). During Gradientinjection, Reagent 1 is pumped by pump (P-1003) through filter (F-1003)and Filtrate Line (1030) into Reagent Line (1020) while during columnwash, Reagent 1 is pumped by pump (P-1003) through Filtrate Line (1030)and into Reagent Line (1031).

Reagent 2 was produced, in Reagent Tank No. 2 (T-1004), from mixingTRIS, added through Reagent Line (1026), and WFI added through WaterLine (1027). Reagent 2 was then filtered by using Reagent No. 2 Pump(P-1004) to pump the Reagent 2 in Tank (T-1004) through Pump Inlet(1021) and Discharge Line (1022), into Reagent No. 2 Filter (F-1004) andback to the tank (T-1004) through Filtrate Line 1032. The pressurecontrol valve (PCV) in Retentate Line (1034) controls the amount ofreagent flow that is allowed to flow past the filter (F-1004) and returndirectly to the tank (T-1004). Reagent Tank No. 2 (T-1004) is a 150gallon tank which includes Sterile Vent Filter (F).

During Gradient injection, Reagent 2 is pumped by pump (P-1004) throughfilter (F-1004) and Filtrate Line (1032) into Reagent Line (1023) whileduring column wash, Reagent 2 is pumped by pump (P-1004) throughFiltrate Line (1032) and into Reagent Line (1033).

Release of phospholipids took place prior to the elution of thehemoglobin, with endotoxins eluting after the hemoglobin peak ofinterest had been collected. Typically, the first portion of the elutinghemoglobin was discarded to waste. Discard of elute to waste from theColumns (C-601A and B) is through Air Operated Valves (AOV 17, AOV 19)and Waste Lines (623, 647) or Waste Lines (636, 637), respectively. Thenthe collection of effluent was begun and continued until the peak or theresponse had been reduced to 20-10% of its peak amplitude. Thisconstituted the fraction which was collected and the fraction ofinterest for purification. Effluent from column (C-601A) flows to Tank(T-601) through Effluent Lines (617, 619, 620) and Air Operated Valves(AOV 14, AOV 17, AOV 22) and then through Combined Effluent Header(602). Similarly, effluent from column (C-601B) flows to Tank (T-601)through Effluent Lines (630, 631, 632) and Air Operated Valves (AOV 21,AOV 18, AOV 22) and then through Combined Effluent Header (602). Thegradient continued to run after hemoglobin collection to removecontaminants from the column prior to commencement of the wash cycle.The wash cycle through Wash Pump (P-603) was comprised of injecting awash into the column used by utilizing firstly Tris/NaCl, supplied fromReagent Supply (1031), then WFI, supplied from Water Line (611) throughAir Operated valve (AOV 3), and then Tris, supplied from Reagent Line(1033) through Air Operated valve (AOV 3), which re-equilibrated thecolumns (C-601A and B) prior to the injection/elution cycle. During thewash cycle, each wash component was pumped by Wash Pump (P-603) throughWash Control Air Operated Valve (AOV 4) and Wash Pump Inlet (638) andthen, was injected into either Column (C-601A, C-601B) through Wash PumpDischarge Line (639), Air Operated Valve (AOV 8) and Wash Supply Lines(640, 641), respectively.

Each Column (C-601A, C-601B) has three in-parallel Bypass Lines (618,621, 622, 633, 634, 635). Bypass Lines (618, 633) will divert someinjection flow around the columns, (C-601A and B) respectively, but donot preclude parallel flow through the columns. Bypass Lines (621, 634)divert all injection flow to columns, (C-601A and B) respectively, tothe outlets of the columns, specifically, Effluent Lines (619, 631).Finally, Bypass Lines (622, 635) also divert all injection flow to thecolumns, (C-601A and B) respectively, to the outlets of the columns,specifically, Waste Lines (639, 637).

The hemoglobin was collected in the Tank (T-601), a 100 gallon vessel,equipped with WFI and Caustic flushing connections, through the combinedinjection pipe (CIP), and Water Line (604) or NaOH Line (605),respectively, Sterile Vent Filter (F) and Nitrogen Supply (606). Thissolution is tetrameric in nature with over 99.9% in the 68,000 Daltonrange as measured by native gel electrophoresis, and high performanceliquid chromatography. The material is pyrogen-free and has amethemoglobin level below 2%. The concentration is 0.2 grams perdeciliter before concentration and can be concentrated to 20 grams perdeciliter.

G. Polymerization (Cross-linking) Reaction

Material was transferred, by means not shown, from the Tank (T-601)through Feed Line (603) to Stage I Cross Link Reactor (R-801) to permitthe concentration of the hemoglobin solution. Stage I Cross Link Reactor(R-801) is a 20 liter vessel equipped with Sterile Filter Vent (F).Concentration of the hemoglobin solution resulted from filtrationthrough the 10,000 molecular weight Stage I Cross Flow Filters (F-803Aand B). During filtration, Stage I Cross Link Pump (P-803) pumpedhemoglobin solution from Reactor (R-801) through Pump Inlet (800) anddischarges hemoglobin solution into filters (F-803A and B) throughDischarge Lines (801, 802, 803). The retentate was recycled to Reactor(R-801) through Filtrate Lines (809, 810). The filtrate was sent towaste through Waste Lines (804, 805, 806, 807, 808). This step wascontinued until a concentration of 7 to 10 grams per deciliter wasobtained and methemoglobin level was less than 1.5%. Duringconcentration, acid can be added to Stage I Cross Link Reactor (R-801)by Acid Pump (P-802) from Acid Supply Line (811) through Discharge Line(812), Acid Filter (F-802) and Filtrate Line (813). In addition, variousreagents can be added by Reagent Feed Pump (P-801) to Stage I Cross LinkReactor (R-801), through Reagent Supply Line (814) through DischargeLine (815), 10K Depyrogenation Filter (F-801) and Filtrate Line (816).This material was bagged and frozen, by means not shown, or pumpeddirectly from Reactor (R-801) through Pump Inlets (810, 817) by theStage II Reactor Feed Pump (P-904) and discharged through Discharge Line(900) into the Stage II Cross-Link Reactor (R-902). Frozen material maybe thawed and fed by gravity directly to the Reactor (R-902), not shown.Stage II Cross Link Reactor (R-902) is a 2 liter vessel equipped withSterile Filter Vent (F).

The processed material was then pumped from Stage II Cross Link Reactor(R-902) by the Stage II Cross Flow Pump (P-905) through 10,000 molecularweight Stage II Cross flow Filter (F-904). Pump (P-905) took a suctionon Reactor (R-902) through Pump Inlets (902, 903) and discharged intoStage II Cross Flow Filter (F-905) through Discharge Lines (904, 907).Permeate recycled to Reactor (R-902) through Filtrate Line (905) andReactor Line (911). The retentate was recycled to the Stage II CrossLink Reactor (R-902). The filtrate was sent to waste through Waste Lines(914, 915, 918, 920). The inside of the reactor was maintained in a lowoxygen environment by drawing a vacuum and blanketing with argon. Thereactor system volume was maintained constant by simultaneously adding,through Pump Inlet (817), Stage II Reactor Feed Pump (P-904) andDischarge Line (900), a sterile pyrogen-free buffer (pH 8.9-9.1) to thereactor through a depyrogenating membrane filter. This buffer wascomprised of sodium, chloride, and potassium. The pH is adjusted withHCl and Tris base. The cross-linking agent (glutaraldehyde) was thenadded to Reactor (R-902) by utilizing Stage II Reactor Feed Pump(P-904), Pump Inlet (817) and Discharge Line (900).

H. Membrane Concentration

Once greater than 50-55% cross-linking, as calculated by gel permeationchromatography, has been achieved, the material was pumped from Stage IICross Link Reactor (R-902) by the Stage II Cross Link Pump (P-905)through the 100,000 molecular weight Stage II Cross Flow Filter (F-905),until about a 25% reduction in fluid volume had been attained. Pump(P-905) took a suction of Reactor (R-902) through Pump Inlets (902, 903)and discharged into filter (F-905) through Discharge Lines (904, 906).Permeate recycled to Reactor (R-902) through Filtrate Line (908) andFermenter Line (912). The filtrate was sent to waste through Waste Lines(916, 917, 919, 920). Electrolytes and pH of the product material wereadjusted during the filtration stage to give a balanced physiologicsolution by additions to the Stage II Cross Link Reactor (R-902)utilizing Stage II Reactor Feed Pump (P-904), Pump Inlet (817) andDischarge Line (900).

The material was then gravity drained to the bag filling machine throughProduct Lines (909, 910, 913). Product was bagged for freezer storage.Analysis of three separate batches of material produced by the aboveprocess, but eliminating optional step E produced cross-linkedhemoglobin solutions having the properties as set forth in the "RESULTS"section of Example IV.

Sodium Hydroxide solution, used throughout this system, was producedfrom mixing sodium hydroxide, added through NaOH Line (1028) andCombined Injection Pipe (1009), and WFI, added through Water Line (1029)and Combined Injection Pipe (1009), in NaOH Loop Tank (T-1002). Thesodium hydroxide solution was then filtered by using NaOH Loop Pump(P-1002) to pump the sodium hydroxide in Tank (T-1002) through PumpInlet (1010) and Discharge Line (1011), into NaOH Loop Filter (F-1002)and back to the tank (T-1002) through Filtrate Line (1012) and RecircLine (1017). The pressure control valve (PCV) in Retentate Line (1036)controls the amount of NaOH flow that is allowed to flow past the filter(F-1002) and return directly to the tank (T-1002). NaOH Loop Tank(T-1002) is a 150 gallon tank which includes Sterile Vent Filter (F).

During NaOH injection into the system, NaOH is pumped by pump (P-1002)through filter (F-1002) and Filtrate Line (1012) into Reagent Lines(1013, 1014, 1015, 1016) and then into Combined Injection Pipes (CIP) bymeans not shown.

Water-For-Injection, used throughout this system, was provided by WFI inWFI Loop Tank (T-1001), which was filled with WFI added through WaterLine (1000). The WFI was then filtered by using WFI Loop Pump (P-1001)to pump the WFI in Tank (T-1001) through Pump Inlet (1001) and DischargeLine (1002), into WFI Loop Filter (F-1001) and back to the tank (T-1001)through Filtrate Line (1003) and Recirc Line (1008). The pressurecontrol valve (PCV) in Retentate Line (1037) controls the amount of WFIflow that is allowed to flow past the filter (F-1001) and returndirectly to the tank (T-1001). WFI Loop Tank (T-1001) is a 300 gallontank which includes Sterile Vent Filter (F).

During WFI injection into the system, WFI is pumped by pump (P-1001)through filter (F-1001) and Filtrate Line (1003) into Water Lines (1004,1005, 1006, 1007) and then into Combined Injection Pipes (CIP) by meansnot shown.

    ______________________________________                                        REFERENCE LIST                                                                ______________________________________                                        P-301  ALBIN SLP 107 P51 B1 Sanitary Lobe-Rotary                                     Pump                                                                   P-302  Same as P-301                                                          P-402  Same as P-301                                                          P-403  Cole Parmer Masterflex Model 7019                                             Peristaltic Pump                                                       P-501  Same as P-301                                                          P-502  Same as P-301                                                          P-601  Same as P-301                                                          P-602  Same as P-301                                                          P-603  Same as P-301                                                          P-905  Albin SLP 110 P51 B1 Sanitary Lobe-Rotary pump                         T-301  25 Gallon 316L Stainless Steel Electro-                                       Polished Interior Fab. by Thermo Electron                                     Wisconsin, Inc.                                                        T-302  Same as T-301                                                          T-401  O-I/Schott 100L Cylindrical Glass Vessel (GER                                 100)                                                                   T-501  Same as P-401                                                          T-502  Same as P-401                                                          T-601  Same as P-401                                                          CT-301 Sharples Model A-16 Type M-3500-520 2HHY CT-302                               SAME AS CT-301                                                         F-401  Stainless steel housing with sanitary pipe connec-                            tions, fitted with millipore 5 sq. ft. filtration                             cassettes                                                              F-402  Same as F-401                                                          F-501A/                                                                              Same as F-401                                                          501B                                                                          F-502A/                                                                              Same as F-401                                                          502B                                                                          F-904  Same as F-401                                                          F-905  same as F-401                                                          C-601A-                                                                              Resistoflex 6" stainless steel TFE lined pipe                          D      and flanges                                                            FR-902 3 liter applicon fermenter (H/D = 2). Agitation:                              2-6 blade paddle impellors (1.5 cm × 1 cm paddles)                      3 cm and 14 cm from the tank bottom, and 4 baffles.                    Bag    Model F-400-X Table Top                                                Filling                                                                              Filling Machine, Cozzi                                                 Machine                                                                              Machine Company.                                                       ______________________________________                                    

EXAMPLE II Molecular Weight Distribution

This study was undertaken to determine molecular weight distribution offinal product. Hemoglobin particles with M.W. more than 1,000,000 mightcause some clinical problems in human and animals. One ul of finalproduct (80 ug of protein) was diluted by 50, and this 50 ul wasinjected into Hewlett-Packard HPLC System. Water Data 740 Module Stationwas used to integrate results.

Since 1980 the classical gel filtration technique employing soft andsemirigid organic gels for protein characterization and purification hasreceived progressively greater competition from high-performancesize-exclusion chromatography (HPSEC). The breakthrough of HPSEC isassociated with the development of highly efficient buffer-compatiblecolumns operating at elevated back pressure. The columns are packed withrigid hydrophilic porous silica gel particles of pre-determined poresize distribution and a derivatized protein-compatible surface. Theproteins elute in the sequence of decreasing molecule weight and size.

Four pyrogen free batches of material with Hb concentrations below wereused in the testing:

    ______________________________________                                        Batch No.        Hb     MET Hb                                                ______________________________________                                        2261             10.3   3.4                                                   2271             10.4   3.8                                                   2311             11.3   7.2                                                   2341             9.0    3.5                                                   ______________________________________                                    

The following protein standards were used:

1. Blue dextran M.W. 2,000,000

2. Aldolase M.W. 158,000

3. Bovine Albumin M.W. 67,000

4. Ovalbumin--M.W. 45,000

5. Ferritin M.W. 540,000

Test System

Columns: One Protein-Pak, 300 sw, Water Associates

Buffer: 0.1M k pH 7.8

Flow rate: 1 ml/min.

HPLC System: Hewlett Packard, 1090 liquid Chromatograph 280 nm Detectorfilter 740 Water Data Module Station

Statistical Analysis

Data was integrated using 740 Water Data Module Station. Before addingglutaraldehyde Hb solution (50 ul) was injected and retention time 9.699was assumed for Hb with 68,000 M.W.

Retention time for blue dextran was around 4.8 min. Retention time forHb solution 4.959 corresponds to M.W. of more than 1,000,000.

Results

Distribution of Molecular Weight:

Wave length:254

Flow rate:1 ml/min

    ______________________________________                                        Retention                                                                     Time      2261    2271       2311   2341                                      ______________________________________                                        4.959     9.4%    7.8%       8.9%   8.5%                                      5.165     8.6%    11.07%     5.2%   3.7%                                      7.362     29.3%   41.9%      33.3%  33.5%                                     8.259     17.8%   15.43%     18.27% 17.5%                                     9.699     34.9%   23.8%      34.33% 36.8%                                     ______________________________________                                    

Conclusions

All 4 batches of material showed very consistent distribution ofmolecular weight from batch to batch. The best Batch was 2271 with 23.8%non-cross-linked material and 7.8% particles with high molecular weight.

The % of cross-linked material for the other 3 batches is:

    ______________________________________                                                   %           % Particles With High                                  Batch No.  Non-X-Linked                                                                              Molecular Weight                                       ______________________________________                                        2261       34.9        9.4                                                    2311       34.3        8.9                                                    2341       36.8        8.5                                                    ______________________________________                                    

At the same time percent of particles with high molecular weight is9.4%, 8.9% and 8.5%.

EXAMPLE III Endotoxin Concentration Determination

The detection of endotoxin concentration within a cross-linked bloodsample was tested by using the Limulus Amebocyte Lysate (LAL) Assaytest. The LAL has been obtained from the extracts of the amebocytes ofthe horseshoe crab. The sample was tested to be positive or negative andwas determined as such against an end-point reaction made by a series ofdilutions of a reference endotoxin. A standard regression curve was madefrom colorimetric readings from the above-mentioned dilutions and theendotoxin content was determined from the interpolation from the curve.

On Jan. 18, 1980 (38 FR 1404), the FDA announced that Limulus AmebocyteLysate derived from amebocytes of the horseshoe crab is now a biologicproduct and can be used in place of rabbits. LAL has proved to be asensitive indicator of bacterial endotoxin or pyrogens within theproduct. Because of its high sensitivity in detecting endotoxins,products can be prevented from being administered to humans which couldcause fever, shock and death if found too high in pyrogens.

Test and Control Articles

Four polymerized blood samples were tested using LAL assay test andfound to be less than 0.01 endotoxin units per ml.

    ______________________________________                                               Batch No.                                                                             Eu/ml                                                          ______________________________________                                               2261    <0.01                                                                 2271    <0.01                                                                 1311    <0.01                                                                 2341    <0.01                                                          ______________________________________                                    

Materials:

1. Glass test tubes depyrogenated by baking in a 180° C. oven for noless than 4 hours, preferably 24 hours

2. Limulus Amebocyte Lysate, Lot #372, Spectrozyme substrate (Cape CodeAssociates)

3. Acetic acid--50% solution, H₂ O used for injection, diluted sample ofpolymerized blood.

EXAMPLE IV Acute Toxicity Determination

Three batches of the blood substitute of the present invention (Hb-I,Hb-II and Hb-III) referred to as hemoglobin below, and human plasmaprotein fraction (PPF), in a controlled study, were used to replaceone-third of estimated blood volume in 4 groups of 6 rabbits each.

This study was undertaken to evaluate the acute toxicity of the bloodsubstitute of the present invention for intravenous administration inrabbits. The study is based in terms of (1) mortality, (2) morbidity,(3) pathophysiologic changes affecting the vital organs, (4) pathologicchanges (gross), and (5) pathologic changes (microscopic). The study isdesigned to compare the effects of 3 batches of the blood substitute(Hb-I, Hb-II, and Hb-III) to those of human Plasma protein Fractions(PPF) following replacement of 1/3 estimated blood volume in 4 groups ofrabbits.

Experimental Model

New Zealand male rabbits of 4.0 Kg body weight; sedated withchlorpromazine, 5 mg/Kg body weight I.M.; restrained.

Instrumented with:

(a) urinary catheter

(b) arterial line (one ear's artery)

(c) venous line (one ear's marginal vein)

(d) needle-electrodes for ECG

(e) thermoprobe (subcutaneous)

    ______________________________________                                        EXPERIMENTAL PROTOCOL                                                         ______________________________________                                        1      Sedation                                                                      Instrumentation                                                               @                                                                             T.sub.1 (baseline) @ and *                                             2      Bleeding                                                                      Infusion of blood substitute                                                  @                                                                             T.sub.2 (15 minutes post-infusion) @ and *                             3      @                                                                             @                                                                             T.sub.3 (1 hour post-infusion) @ and *                                        @                                                                      4      @                                                                             @                                                                             @                                                                             @                                                                      5      @                                                                             @                                                                             @                                                                             T.sub.4 (3 hours post-infusion) @ and *; cannulae and                         electrodes removed; Animal returned to the cage                        24     T.sub.5 (24 hours post-infusion) @ and * necropsy                      ______________________________________                                         @ = measurement of hemodynamic parameters                                     * = blood and urine samples                                              

Anesthesia. The animals were sedated with chlorpromazine 0.5 mg/kg bodyweight injected I.M., restrained in a metal rabbit-holder and allowed tospontaneously breathe room air. Body temperature was maintained byplacing the animal on an electric heating pad.

Instrumentation. Plastic cannulae (22-gauge) were inserted into bothears' central arteries (one connected to a pressure transducer tomonitor the arterial blood pressure and the other used for removal andsampling of arterial blood) and the ear veins for the infusion ofhemoglobin solution. Needle electrodes were placed in the limbs tomonitor the electrocardiogram. A catheter was inserted into the urinarybladder for the measurement of urinary output and the collection ofurine samples. A temperature probe was inserted subcutaneously tomonitor body temperature.

Procedure. Arterial blood was removed in the amount of 20 ml/kg bodyweight (approximately 1/3 of estimated blood volume) and this amount wasimmediately replaced with an equal amount of hemoglobin solution. Allblood subsequently removed for laboratory testing was replaced withhemoglobin, 1:1 v:v. The animal was closely observed for a period of 3hours (time necessary for the completion of the pyrogenicity test).During this interval, additional doses of chlorpromazine wereadministered as necessary to maintain the animal sedated, and anintravenous infusion of 5% dextrose in 1/4 normal saline wasadministered, 15 ml/Kg body weight/hour, to replace water losses.

Electrocardiogram, blood pressure and body temperature were monitoredcontinuously and recorded at 15-minute intervals. Urinary output wasrecorded at 30-minute intervals. Blood samples were taken at baseline,and 15 minutes, 1 hour and 3 hours after completion of the hemoglobininfusion. All monitoring lines were then disconnected and the animal wasreturned to its cage, where it was allowed water "ad libitum".Additional blood samples were taken at 6, 12 and 24 hours, with theanimal again restrained in the rabbit-holder and using the ear arteries.After 24 hours, the animal was killed with an overdose of pentobarbitaland a complete necropsy was carried out. Special attention was paid tothe possible presence of hemoglobin pigment in the body cavities,including the anterior chamber of the eye. Sections were taken all oforgans of histologic examination.

The following tests were carried out on each blood sample:

1) Complete CBC, including platelet count (Coulter Counter),

2) PTT (MLA 700); fibrinogen; fibrin split products

3) Electrolytes (sodium, potassium, chloride and bicarbonate) (ASTRAApparatus),

4) Alakaline P-ase, LDH, SGOT and SGPT (ASTRA Apparatus)

5) BUN and creatinine (ASTRA Apparatus)

6) Osmolarity (Vapor pressure)

7) Plasma hemoglobin concentration (Benzidine)

8) Arterial Blood Gases (IL pH/Blood Gas Analyzer)

9) Total Hb, Oxy-Hb, CO-Hb, Met-Hb and O₂ Content (IL 282 Co-oximeter)

10) P₅₀ (Aminco Hem-O-Scan)

Urine was tested for:

1) hemoglobin concentration (Benzidine)

2) creatinine (ASTRA Apparatus)

3) sodium and potassium (ASTRA Apparatus)

By the time collection of urine and the determination of plasma andurine creatinine, a Creatinine Clearance Test was carried out at the3-hour interval post-hemoglobin infusion.

The data will be tabulated as shown in the following tables.

Data Evaluation. The data obtained from the 6 animals in each group wastabulated as Mean Values±Standard Errors. The statistical significanceof changes related to time was evaluated by analysis of variance (TableIII). Comparisons between the various groups of animals was made usingStudent's T-test for paired data (Table IV). The raw data for compilingTables III and IV is presented in Tables I and II.

    ______________________________________                                        METHODS                                                                       ______________________________________                                        (A)  Mortality                                                                (B)  Morbidity                                                                Attention was paid to the development of the following                        manifestations:                                                               1 =  anaphylactic shock                                                       2 =  seizures or development of neurologic deficits                           3 =  bronchospasm or pulmonary edema (immediate effects)                      4 =  fever                                                                    5 =  hemoglobinuria                                                           6 =  hyphema                                                                  7 =  lack of normal activity                                                  8 =  depression of normal functions (eating and drink-                             ing) at 24 hours                                                         (C)  Pathophysiologic changes                                                 These changes were studied according to the following scheme:                 1.   Clinical observations:                                                        a. body weight                                                                b. body temperature                                                           c. heart rate                                                                 d. arrhythmias                                                                e. blood pressure (systolic)                                                  f. blood pressure (diastolic)                                                 g. urinary output                                                        2.   Laboratory data reflecting respiratory                                        function:                                                                     a. arterial blood pH                                                          b. PaO.sub.2                                                                  c. PaCO.sub.2                                                                 d. P.sub.50                                                              3.   Hematology (Coulter);                                                         a. hematocrit                                                                 b. hemoglobin                                                                 c. WBC                                                                        d. platelets                                                             4.   Coagulation:                                                                  a. fibrinogen                                                                 b. fibrin split products                                                      c. P.T.                                                                  5.   Liver function:                                                               a. total bilirubin                                                            b. SGOT                                                                       c. LDH                                                                        d. SGPT                                                                  6.   Renal function:                                                               a. BUN                                                                        b. serum creatinine                                                           c. serum electrolytes                                                         d. serum osmolality                                                      ______________________________________                                    

(D) Gross Pathology

At necropsy, attention was focused on hemoglobin extravasation: into theanterior chamber of the eye, the pericardium, the pleurae and theperitoneum.

Heart, lungs, liver, spleen and kidneys were examined for gross signs ofedma, congestion, hemorrhage and infarction.

(E) Histopathology

Section of heart, lungs, liver, spleen and kidneys were processed for,and examined by, light microscopy.

A method of grading the histopathologic changes was developed in orderto perform a statistical analysis of the data.

The main change seen in the heart was represented by focal areas ofmyocardial contracture. Each focal area found on a transverse section ofthe left ventricle was assigned a 1+grade.

In the lungs, pathology was also patchy. The main changes wererepresented by interstitial edema and cell infiltration ("interstitialpneumonia"). Grading was carried out from an overall picture of tissueinvolvement, as well as from the severity of changes observed in eachinvolved area, developing a scale of 1-to-4.

In the liver, the main alterations were represented by congestion and bycentrolobular vacuolization. These changes were graded on a scale of1-to-4 on the basis of both, the number of lobules involved and theextent of vacuolization starting from the centrolobular venule.

In the spleen, congestion was the main finding.

In the kidney, no glomerular alteration and no tubular necrosis orblockage by pigment casts was found. The main alteration was representedby a vacuolization of the tubular epithelium, starting at thesubcapsular area and extending from there toward the corticomedullaryjunction. The degree of this extension was graded on a scale of 1-to-4.

Statistical Analysis

Analysis of the data was carried out using two tests:

(a) Analysis of variance to study changes occurring in each group ofanimals at various time intervals; and

(b) Student's t-test for paired data to study the changes occurring ateach time interval in the various groups of animals.

Results of statistical analyses performed are presented in Tables IIIand IV.

                                      TABLE I                                     __________________________________________________________________________    T-1 [B.L.] T-2 [15 min.]                                                                          T-3 [1 hr.]                                                                            T-4 [3 hrs.]                                                                           T-5 [24 hrs.]                           M ± SD  M ± SD                                                                              M ± SD                                                                              M ± SD                                                                              M ± SD                               __________________________________________________________________________    1. BODY WEIGHT [gm]                                                           PPF  4166 ± 262.4                                                                     --       --        4260 ± 309.8                                                                        4170 ± 292.0                        Hb I                                                                               4350 ± 125.8                                                                     --       --        4510 ± 125.0                                                                        4366 ± 110.5                        Hb II                                                                              4150 ± 236.2                                                                     --       --        4285 ± 226.8                                                                        4150 ± 211.9                        Hb III                                                                             3966 ± 47.0                                                                      --       --        4092 ± 84.0                                                                         4014 ± 51.0                         2. BODY TEMPERATURE [°C.]                                              PPF  38.7 ± 0.42                                                                       38.5 ± 0.44                                                                         3.85 ± 0.46                                                                         38.5 ± 0.47                                                                         38.6 ± 0.45                         Hb I                                                                               38.8 ± 0.38                                                                       3.8 ± 0.38                                                                          38.9 ± 0.46                                                                         38.9 ± 0.58                                                                         39.0 ± 0.58                         Hb II                                                                              39.1 ± 0.09                                                                       38.8 ± 0.37                                                                         38.9 ± 0.63                                                                         38.9 ± 0.69                                                                         38.9 ± 0.18                         Hb III                                                                             39.4 ± 0.40                                                                       39.0 ± 0.50                                                                         39.1 ± 0.46                                                                         38.9 ± 0.68                                                                         39.1 ± 0.71                         3. HEART RATE [Beats/min]                                                     PPF   220 ± 21.9                                                                        200 ± 13.20                                                                         195 ± 21.23*                                                                        197 ± 16.43*                                                                        198 ± 5.03*                        Hb I                                                                                230 ± 5.77                                                                        212 ± 8.49**                                                                        218 ± 8.97*c                                                                        225 ± 7.63b                                                                         222 ± 12.13b                       Hb II                                                                               220 ± 17.71                                                                       200 ± 11.60                                                                         199 ± 15.60*                                                                        203 ± 18.97                                                                         203 ± 9.42                         Hb III                                                                              211 ± 24.00                                                                       190 ± 33.00                                                                         196 ±  33.00                                                                        213 ± 39.00b                                                                        202 ± 10.00                        4. BLOOD PRESSURE (SYSTOLIC) [mm Hg]                                          PPF   100 ± 6.32                                                                       95.8 ± 14.06                                                                         95 ± 10.00                                                                         96.7 ± 5.18                                                                          96 ± 4.18                          Hb I                                                                              104.5 ± 14.8                                                                      113.3 ± 12.13c                                                                      109.5 ± 10.22c                                                                      108.3 ± 10.27c                                                                      108.3 ± 10.27c                       Hb II                                                                              95.8 ± 5.33                                                                      111.6 ± 15.72*c                                                                     105.8 ± 11.33                                                                       106.7 ± 12.13                                                                         105 ± 8.16                         Hb III                                                                             97.3 ± 0.21                                                                       95.2 ± 7.00                                                                        102.3 ± 9.59                                                                         90.8 ± 7.86                                                                         88.3 ± 6.20*                        5. BLOOD PRESSURE (DIASTOLIC) [mm Hg]                                         PPF  60.8 ± 9.17                                                                       60.8 ± 14.63                                                                        60.8 ± 9.70                                                                         62.5 ± 6.89                                                                         65.0 ± 4.08                         Hb I                                                                               70.8 ± 15.91                                                                      80.0 ± 14.71c                                                                       77.5 ± 14.92c                                                                       77.5 ± 13.96                                                                        74.2 ± 12.72                        Hb II                                                                              60.0 ± 8.16                                                                       75.8 ± 15.11*c                                                                      73.3 ± 11.05*                                                                       70.8 ± 9.75                                                                         67.5 ± 6.92                         Hb III                                                                             64.2 ± 3.43                                                                       69.2 ± 5.32                                                                         73.8 ± 7.53*                                                                        63.3 ± 7.60                                                                         61.7 ± 6.20                         6. URINARY OUTPUT [ml/30 min.]                                                PPF  2.60 ± 0.49                                                                       0.0 ± 0.00***                                                                       3.00 ± 0.61                                                                         3.10 ± 0.63                                                                         3.00 ± 0.53                         Hb I                                                                               3.11 ± 0.29                                                                       0.0 ± 0.00***                                                                       6.66 ± 1.72**b                                                                      5.66 ± 1.59**c                                                                      4.63 ± 0.96**c                      Hb II                                                                              3.00 ± 0.57                                                                       0.0 ± 0.00***                                                                       5.30 ± 1.59*b                                                                       5.10 ± 1.64*                                                                        4.25 ± 1.46                         Hb III                                                                             5.66 ± 1.99c                                                                      6.50 ± 4.63*a                                                                      10.80 ± 2.90**a                                                                     11.30 ± 4.20**a                                                                      5.40 ± 1.26b                        7. ARTERIAL BLOOD pH [UNITS]                                                  PPF  7.33 ± 0.04                                                                       7.34 ± 0.05                                                                         7.33 ± 0.03                                                                         7.33 ± 0.03                                                                         7.39 ± 0.13                         Hb I                                                                               7.43 ± 0.10                                                                       7.36 ± 0.13                                                                         7.37 ± 0.11                                                                         7.37 ± 0.10                                                                         7.38 ± 0.06                         Hb II                                                                              7.36 ± 0.03                                                                       7.36 ± 0.03                                                                         7.34 ± 0.12                                                                         7.42 ± 0.11                                                                         7.36 ± 0.04                         Hb III                                                                             7.28 ± 0.03                                                                       7.30 ± 0.02                                                                         7.28 ± 0.05                                                                         7.26 ± 0.06                                                                         7.31 ± 0.03                         8. PaO.sub.2 [mm Hg]                                                          PPF  69.9 ± 8.29                                                                       70.8 ± 11.72                                                                        62.0 ± 4.67*                                                                        59.2 ± 8.09*                                                                        69.2 ± 5.88                         Hb I                                                                               74.1 ± 16.7                                                                       67.5 ± 8.54                                                                         73.7 ± 10.2c                                                                        71.5 ± 11.3c                                                                        60.6 ± 7.78                         Hb II                                                                              63.8 ± 2.12                                                                       69.2 ± 6.14                                                                         81.6 ± 9.66***b                                                                     79.5 ± 5.88***a                                                                     76.9 ± 8.77***                      Hb III                                                                             63.3 ± 12.2                                                                        76.3 ± 14.4                                                                        70.7 ± 7.30c                                                                        88.1 ± 18.3*b                                                                       69.9 ± 13.0c                        9. PaCO.sub.2 [mm Hg]                                                         PPF  29.8 ± 3.24                                                                       31.2 ± 2.77                                                                         33.7 ± 2.98*                                                                        34.8 ± 2.11*                                                                        31.9 ± 5.15                         Hb I                                                                               29.8 ± 3.08                                                                       33.0 ± 5.27                                                                         34.4 ± 4.59*                                                                        32.6 ± 5.33                                                                         32.5 ± 5.12                         Hb II                                                                              28.8 ± 5.37                                                                       31.0 ± 1.89                                                                         31.9 ± 3.47                                                                         30.0 ± 4.30c                                                                        31.2 ± 2.01                         Hb III                                                                             32.8 ± 2.56                                                                       27.6 ± 0.62**c                                                                      29.9 ± 2.50c                                                                        33.0 ± 3.49                                                                         32.4 ± 1.45                         10. BLOOD P.sub.50 [mmHg]                                                     PPF  34.0 ± 0.61                                                                       33.7 ± 1.75                                                                         33.8 ± 3.01                                                                         34.0 ± 1.80                                                                         34.0 ± 1.80                         Hb I                                                                          Hb II                                                                              33.7 ± 0.87                                                                       29.7 ± 0.63***b                                                                     30.8 ± 0.47**                                                                       32.7 ± 0.62                                                                         33.0 ± 1.41                         Hb III                                                                             32.4 ± 1.45                                                                       27.7 ± 1.43***a                                                                     29.5 ± 1.29**b                                                                      30.8 ± 1.24c                                                                        31.6 ± 0.41c                        11. COLLOID OSMOTIC PRESSURE (COP) [mmHg]                                     PPF  18.2 ± 0.53                                                                       19.2 ± 0.06*                                                                        19.3 ± 0.77*                                                                        18.8 ± 0.60                                                                         18.3 ± 0.59                         Hb I                                                                          Hb II                                                                              17.9 ± 0.49                                                                       19.5 ± 0.55**                                                                       19.5 ± 1.17*                                                                        19.3 ± 1.21                                                                         18.5 ± 0.63                         Hb III                                                                             17.5 ± 0.07                                                                       19.5 ± 0.77***                                                                      29.5 ± 1.29***                                                                      18.5 ± 0.59**                                                                       17.9 ± 0.38*                        12. PLASMA Hb [g/dl]                                                          PPF  0.00 ± 0.00                                                                       0.00 ± 0.00                                                                         0.00 ± 0.00                                                                         0.00 ± 0.00                                                                         0.00 ± 0.00                         Hb I                                                                          Hb II                                                                              0.00 ± 0.00                                                                       3.82 ± 0.71***a                                                                     3.50 ± 0.68***a                                                                     3.10 ±  0.40***a                                                                    2.10 ± 0.51***                      Hb III                                                                             0.00 ± 0.00                                                                       3.16 ± 0.78***a                                                                     2.70 ± 0.79***a                                                                     2.60 ± 0.70***a                                                                     2.10 ± 0.66***                      13. WBC [× 10.sup.9 ]                                                   PPF  6.27 ± 2.13                                                                       6.48 ± 1.16                                                                         7.25 ± 2.15                                                                         6.58 ± 1.53                                                                         6.55 ± 1.20                         Hb I                                                                               4.86 ± 0.85                                                                       3.56 ± 0.43*a                                                                       5.70 ± 1.53                                                                         6.48 ± 2.11*                                                                        8.60 ± 1.24**                       Hb II                                                                              4.88 ± 0.87                                                                       4.70 ± 1.94                                                                         5.05 ± 1.29                                                                         4.78 ± 0.69c                                                                        6.22 ± 1.45                         Hb III                                                                             5.83 ± 1.38                                                                       3.17 ± 0.84**a                                                                      2.98 ± 1.06**b                                                                      4.22 ± 2.38                                                                        11.50 ± 6.84                         14. RBC [× 10.sup.12 ]                                                  PPF  5.10 ± 0.42                                                                       3.75 ± 0.42***                                                                      3.90 ± 0.27***                                                                      3.80 ± 0.39***                                                                      3.79 ± 0.32**                       Hb I                                                                               4.86 ± 0.23                                                                       3.78 ± 0.30***                                                                      3.83 ± 0.30***                                                                      3.79 ± 0.33***                                                                      3.96 ± 0.24**                       Hb II                                                                              5.32 ± 0.33                                                                       3.86 ± 0.19***                                                                      3.86 ± 0.17***                                                                      3.87 ± 0.22***                                                                      4.18 ± 0.64**                       Hb III                                                                             5.49 ± 0.55                                                                       4.44 ± 0.33**c                                                                      4.47 ± 0.37**c                                                                      4.18 ± 0.63**                                                                       3.87 ± 0.39**                       15. HEMOGLOBIN [g/dl]                                                         PPF  11.6 ± 0.82                                                                       8.4 ± 0.93***                                                                       8.8 ± 0.59***                                                                       8.6 ± 0.72***                                                                       8.7 ± 0.72***                       Hb I                                                                               11.3 ± 0.75                                                                       11.1 ± 0.30a                                                                        11.2 ± 0.69a                                                                        10.7 ± 0.42a                                                                        11.1 ± 0.23a                        Hb II                                                                              11.6 ± 0.85                                                                       11.2 ± 0.68a                                                                        11.0 ± 0.54a                                                                        10.6 ± 0.30a                                                                        10.2 ± 3.20a                        Hb III                                                                             12.1 ± 1.25                                                                       12.1 ± 1.00a                                                                        12.0 ± 0.96a                                                                        11.3 ± 1.04a                                                                        10.0 ±  0.91*a                      16. HEMATOCRIT [%]                                                            PPF  33.7 ± 2.85                                                                       24.6 ± 2.77***                                                                      25.5 ± 1.77***                                                                      24.9 ± 2.01***                                                                      24.9 ± 1.98***                      Hb I                                                                               32.9 ± 2.14                                                                       25.5 ± 2.32***                                                                      25.8 ± 1.64***                                                                      25.5 ± 1.44***                                                                      28.1 ± 2.01*                        Hb II                                                                              34.2 ± 2.23                                                                       24.7 ± 1.14***                                                                      24.8 ± 0.82***                                                                      24.8 ± 1.25***                                                                      26.8 ± 4.07**                       Hb III                                                                             35.4 ± 2.69                                                                       28.4 ± 2.24**b                                                                      28.5 ± 2.25**c                                                                      27.4 ± 2.30***                                                                      25.3 ± 2.84***                      17. PLATELETS [× 10.sup.9 ]                                             PPF   388 ± 54.5                                                                        300 ± 45.6*                                                                         336 ± 51.8**                                                                        314 ± 39.8*                                                                         376 ± 36.2                         Hb I                                                                                363 ± 48.7                                                                        248 ± 18.7***c                                                                      236 ± 43.5**b                                                                       256 ± 24.9**c                                                                       367 ±  111.2                       Hb II                                                                               411 ± 86.5                                                                        241 ± 70.9**                                                                        263 ± 78.9**                                                                        292 ± 69.8*                                                                         350 ± 69.2                         Hb III                                                                              461 ± 63.0                                                                        264 ± 111.0**                                                                       281 ± 97.0**                                                                        274 ± 71.0**                                                                        402 ± 158.0                        18. FIBRINOGEN [mg/dl]                                                        PPF   226 ± 31.8                                                                        165 ± 19.7**                                                                        180 ± 11.5**                                                                        191 ± 21.5*                                                                         334 ± 32.6***                      Hb I                                                                                238 ± 35.3                                                                        147 ± 25.5*                                                                         140 ± 23.8***b                                                                      139 ± 25.7***b                                                                      291 ± 112.7                        Hb II                                                                               240 ± 83.4                                                                        142 ± 28.9*                                                                         131 ± 28.2*b                                                                        125 ± 19.3*b                                                                        294 ± 96.3                         Hb III                                                                              234 ± 39.0                                                                        152 ± 43.0*                                                                         138 ± 38.0**b                                                                       126 ± 36.0**b                                                                        355 ± 149.0                       19. FIBRIN SPLIT PROD. [mcg/ml] [0 = <10 mcg/ml,                              1 = >10 < 40 mcg/ml, 2 = >40 mcg/ml]                                          PPF  0.00 ± 0.0                                                                        0.00 ± 0.0                                                                          0.16 ± 0.37                                                                         0.00 ± 0.0                                                                          0.00 ± 0.0                          Hb I                                                                               0.16 ± 0.37                                                                       1.0 ± 0.57*b                                                                        1.16 ± 0.68*c                                                                       1.16 ± 0.37**b                                                                      0.75 ± 0.42**b                      Hb II                                                                              0.00 ± 0.0                                                                        0.50 ± 0.50*c                                                                       0.66 ± 0.74                                                                         0.50 ± 0.50                                                                         0.50 ± 0.50                         Hb III                                                                             0.00 ± 0.0                                                                        0.00 ± 0.0                                                                          0.00 ± 0.0                                                                          0.16 ± 0.37                                                                         0.16 ± 0.37                         20. PROTHROMBIN TIME [sec]                                                    PPF  10.8 ± 2.11                                                                       11.2 ± 1.64                                                                         9.25 ± 2.00                                                                         9.4 ± 1.00                                                                          8.0 ± 0.32*                         Hb I                                                                               10.7 ± 1.32                                                                       11.1 ± 2.33                                                                         11.5 ± 2.64                                                                         12.2 ± 2.72                                                                         10.0 ± 1.27c                        Hb II                                                                              9.6 ± 0.72                                                                        9.4 ± 0.51c                                                                         10.6 ± 2.21                                                                         9.4 ± 0.36                                                                          8.4 ± 0.48*                         Hb III                                                                             10.1 ± 1.72                                                                       11.8 ± 2.52                                                                         11.2 ± 2.15                                                                         11.0 ± 1.36                                                                         9.3 ± 2.54                          21. TOTAL BILIRUBIN [mg/dl]                                                   PPF  0.33 ± 0.05                                                                       0.20 ± 0.08                                                                         0.23 ± 0.05                                                                         0.23 ± 0.09                                                                         0.27 ± 0.11                         Hb I                                                                          Hb II                                                                              0.17 ± 0.04                                                                       0.13 ± 0.04                                                                         0.33 ± 0.26                                                                         0.17 ± 0.09                                                                         0.63 ± 0.20                         Hb III                                                                             0.18 ± 0.13                                                                       0.17 ± 0.10                                                                         0.28 ± 0.20                                                                         0.32 ± 0.26                                                                         0.42 ± 0.19*                        22. SGOT (AST) [Iu/L]                                                         PPF  39.6 ± 4.96                                                                       38.0 ± 4.53*                                                                        29.7 ± 5.85*                                                                        30.0 ±  3.66*                                                                       69.7 ± 7.49***                      Hb I                                                                               28.3 ± 11.32                                                                      30.0 ± 8.16                                                                         35.0 ± 5.00                                                                         25.1 ± 8.37                                                                        108.3 ± 57.6c                        Hb II                                                                              39.5 ± 16.3                                                                       38.3 ± 2.35                                                                         36.6 ± 4.70                                                                         58.7 ± 18.7c                                                                        38.0 ± 8.36a                        Hb III                                                                             41.8 ± 15.0                                                                       38.3 ± 8.40                                                                         37.5 ± 4.78                                                                         40.0 ± 7.10c                                                                         493 ± 305.9**a                     23. L D H [Iu/L]                                                              PPF  69.0 ± 12.0                                                                       50.8 ± 5.83**                                                                       60.0 ± 14.0                                                                         52.6 ± 9.63*                                                                       135.0 ± 35.5***                      Hb I                                                                               66.0 ± 34.1                                                                       60.8 ± 19.2                                                                         78.0 ± 6.00c                                                                         112 ± 32.9*b                                                                       73.3 ± 33.1c                        Hb II                                                                              61.0 ± 39.3                                                                       69.6 ± 15.5                                                                         67.5 ± 10.2                                                                         70.6 ± 24.5                                                                        109.2 ± 17.9                         Hb III                                                                             52.7 ± 12.3                                                                       46.0 ± 19.5                                                                         44.7 ± 8.10                                                                         58.0 ± 9.27                                                                        927.0 ± 477**c                       24. SGPT (ALT) [uI/L]                                                         PPF  46.2 ± 8.67                                                                       31.5 ± 7.99*                                                                        33.2 ± 8.82*                                                                        32.5 ± 8.48*                                                                        60.5 ± 5.44*                        Hb I                                                                               48.6 ± 9.92                                                                       43.3 ± 2.35                                                                         46.6 ± 2.35c                                                                        41.0 ± 13.5                                                                         48.8 ± 16.8                         Hb II                                                                              46.3 ± 20.2                                                                       48.8 ± 25.2                                                                         40.0 ± 4.08                                                                         39.0 ± 7.34                                                                         43.2 ± 23.1                         Hb III                                                                             41.2 ± 11.6                                                                       43.6 ± 10.6                                                                         37.0 ± 9.27                                                                         41.2 ± 4.13                                                                        191.5 ± 119**c                       25. B U N [mg/dl]                                                             PPF  16.2 ± 2.13                                                                       16.2 ± 3.37                                                                         14.5 ± 2.43                                                                         14.2 ± 2.40                                                                         13.5 ± 2.08                         Hb I                                                                               21.0 ± 2.82c                                                                      21.8 ± 2.88c                                                                        21.5 ± 2.93c                                                                        21.8 ± 3.33c                                                                        20.6 ± 4.41b                        Hb II                                                                               22.5 ± 5.50                                                                      22.8 ± 5.39c                                                                        22.3 ± 4.81c                                                                        20.8 ± 5.07c                                                                        21.0 ± 2.81b                        Hb III                                                                             17.0 ± 1.52                                                                       17.2 ± 1.67                                                                         17.3 ± 1.48c                                                                        16.2 ± 1.34                                                                         20.7 ± 4.49b                        26. SERUM CREATININE [mg/dl]                                                  PPF  1.25 ± 0.18                                                                       1.23 ± 0.18                                                                         1.05 ± 0.10                                                                         1.07 ± 1.16                                                                         1.10 ± 0.12                         Hb I                                                                               1.43 ± 0.14                                                                       2.26 ± 1.05                                                                         1.85 ± 0.91c                                                                        1.88 ± 0.44b                                                                        1.84 ± 0.46c                        Hb II                                                                              1.50 ± 0.30                                                                       1.31 ± 0.65                                                                         1.24 ± 0.57                                                                         1.64 ± 0.31c                                                                        1.32 ± 0.42                         Hb III                                                                             1.38 ± 1.17                                                                       2.15 ± 1.13                                                                         2.32 ± 0.90c                                                                        2.60 ± 1.35*c                                                                       2.2 ± 0.42c                         27. SERUM SODIUM [mEq/l]                                                      PPF 137.0 ± 2.19                                                                      139.0 ± 2.86                                                                        140.5 ± 4.08                                                                        141.0 ±  4.31                                                                       145.0 ± 1.25***                      Hb I                                                                              137.2 ± 2.26                                                                      134.8 ± 3.23c                                                                       139.5 ± 10.7                                                                        136.6 ± 3.72                                                                        140.8 ± 4.31c                        Hb II                                                                             134.8 ± 2.11                                                                      131.3 ± 4.18c                                                                       133.0 ± 1.73b                                                                       135.5 ± 8.21                                                                        140.0 ± 4.05*c                       Hb III                                                                            130.0 ± 3.50c                                                                     129.0 ± 5.10b                                                                       128.6 ± 5.10a                                                                       129.8 ± 4.00b                                                                       138.0 ± 3.50*c                       28. SERUM POTASSIUM [mEq/l]                                                   PPF  3.45 ± 0.26                                                                       3.30 ± 0.30                                                                         3.50 ± 0.27                                                                         3.27 ± 0.28                                                                         4.35 ± 0.35***                      Hb I                                                                               3.71 ± 0.48                                                                       3.78 ± 0.35c                                                                        4.23 ± 1.64                                                                         3.50 ± 0.43                                                                         3.84 ± 0.32                         Hb II                                                                              3.61 ± 0.48                                                                       3.42 ± 0.34                                                                         3.45 ± 0.26                                                                         3.53 ± 0.39                                                                         4.32 ± 0.17*                        Hb III                                                                             3.60 ± 0.41                                                                       3.33 ± 0.43                                                                         3.15 ± 0.29                                                                         3.23 ± 0.27                                                                         3.60 ± 0.79                         29. SERUM CHLORIDE [ mEq/l]                                                   PPF 101.2 ± 2.71                                                                      104.7 ± 2.50*                                                                       107.5 ± 3.56**                                                                      110.0 ± 2.53***                                                                     104.7 ± 2.75                         Hb I                                                                              105.1 ± 4.56                                                                      105.8 ± 3.34                                                                        105.8 ± 4.22                                                                        105.2 ± 3.34c                                                                       105.6 ± 3.20                         Hb II                                                                             102.3 ± 2.75                                                                      103.6 ± 2.81                                                                        103.8 ± 1.95                                                                        106.3 ± 6.79                                                                        103.7 ± 2.16                         Hb III                                                                             97.0 ± 3.60                                                                       95.8 ± 5.50                                                                         98.0 ± 3.50b                                                                        97.5 ± 6.00b                                                                       102.0 ± 5.60                         30. SERUM BICARB. [mEq/l]                                                     PPF  22.2 ± 2.64                                                                       22.7 ± 2.80                                                                         23.8 ± 1.60                                                                         24.3 ± 1.37                                                                         26.2 ± 4.92                         Hb I                                                                               22.6 ± 1.69                                                                       22.2 ± 2.67                                                                         22.4 ± 2.13                                                                         22.0 ± 2.00c                                                                        22.8 ± 1.47                         Hb II                                                                              23.5 ± 2.50                                                                       22.5 ± 2.69                                                                         22.5 ± 2.21                                                                         20.2 ± 2.67b                                                                        22.2 ± 2.05                         Hb III                                                                             22.3 ± 1.90                                                                       19.5 ± 2.50                                                                         18.5 ± 3.25*b                                                                       20.0 ± 2.40b                                                                        20.6 ± 2.80                         31. SERUM OSMOLALITY [mOsm/kg]                                                PPF 304.4 ± 11.3                                                                      299.5 ± 11.3                                                                        290.5 ± 8.65*                                                                       292.8 ± 9.91                                                                        293.7 ± 3.21*                        Hb I                                                                              300.0 ± 5.38                                                                      299.5 ± 6.20                                                                        296.5 ± 5.70                                                                        295.2 ± 6.88                                                                        296.2 ± 5.11                         Hb II                                                                             309.0 ± 18.8                                                                      299.0 ± 7.34                                                                        300.2 ± 11.0                                                                        294.6 ± 10.5                                                                        297.2 ± 5.35                         Hb III                                                                            301.8 ± 8.80                                                                      281.4 ± 6.80**c                                                                     279.6 ± 8.20**                                                                      279.0 ± 6.50**c                                                                     284.6 ± 6.70*                        __________________________________________________________________________     M: ARITHMETIC MEAN                                                            ± SD: STANDARD DEVIATION                                                   DIFFERENCES BETWEEN T1 TO T2, T3, T4, T5:                                     ***  significant difference P < 0.001                                         **  significant difference P < 0.01                                           *  significant difference P < 0.05                                            DIFFERENCES BETWEEN THE GROUPS:                                               PPFHb I                                                                       PPFHb II                                                                      PPFHb III:                                                                    a  significant difference P < 0.001                                           b  significant difference P < 0.01                                            c  significant difference P < 0.05                                            n = 6 rabbits                                                            

                                      TABLE II                                    __________________________________________________________________________    1. HISTOPATHOLOGY [after 24 hours]                                                   HEART     LUNGS KIDNEYS     LIVER SPLEEN                                      M ± SD M ± SD                                                                           M ± SD   M ± SD                                                                           M ± SD                            __________________________________________________________________________    PPF    1.00 ± 0.00                                                                          1.40 ± 0.48                                                                      1.60 ± 0.48                                                                            1.40 ± 0.48                                                                      1.00 ± 0.00                       Hb I   1.50 ± 0.50                                                                          2.00 ± 0.57                                                                      1.83 ± 0.36                                                                            1.50 ± 0.76                                                                      1.00 ± 0.00                       Hb II  1.66 ± 0.74                                                                          1.83 ± 0.37                                                                      2.16 ± 0.37                                                                            1.83 ± 0.68                                                                      1.00 ± 0.00                       Hb III 1.33 ± 0.46                                                                          1.83 ± 0.37                                                                       2.66 ± 0.46**                                                                         2.00 ± 0.57                                                                      1.00 ± 0.00                       __________________________________________________________________________        T-1    T-2     T-3     T-4     T-5     T-7                                    M ± SD                                                                            M ± SD                                                                             M ± SD                                                                             M ± SD                                                                             M ± SD                                                                             M ± SD                          __________________________________________________________________________    1. BODY WEIGHT [gm]                                                           PPF  4166 ± 262.4                                                                     --      --       4260 ± 309.8                                                                       4170 ± 292.0                                                                      --                                 Hb IV                                                                              4117 ± 146.2                                                                     --      --       4185 ± 147.0                                                                      --      --                                 2. BODY TEMPERATURE [°C.]                                              PPF  38.7 ± 0.42                                                                       38.5 ± 0.44                                                                        38.5 ± 0.46                                                                        38.5 ± 0.47                                                                        38.6 ± 0.45                                                                       --                                 Hb IV                                                                              39.0 ± 0.12                                                                       38.9 ± 0.34                                                                        38.8 ± 0.52                                                                        38.8 ± 0.44                                                                        39.0 ± 0.17                                                                       --                                 3. HEART RATE [Beats/min]                                                     PPF   220 ± 21.9                                                                        200 ± 13.20                                                                        195 ± 21.23*                                                                       197 ± 16.43*                                                                       198 ± 5.03*                                                                      --                                 Hb IV                                                                               232 ± 8.97                                                                        191 ± 20.08**                                                                      197 ± 15.96**                                                                      189 ± 20.49**                                                                      207 ± 17.94*                          4. BLOOD PRESSURE (SYSTOLIC) [mm Hg]                                          PPF    100 ± 6.32                                                                      95.8 ± 14.06                                                                        95 ± 10.00                                                                        96.7 ± 5.18                                                                         96 ± 4.18                                                                        --                                 Hb IV                                                                               88 ± 3.26b                                                                       92.0 ± 4.47                                                                        89.6 ± 10.02                                                                      90.03 ± 4.67                                                                         88 ± 0.00c                                                                       --                                 5. BLOOD PRESSURE (DIASTOLIC) [mm Hg]                                         PPF  60.8 ± 9.17                                                                       60.8 ± 14.63                                                                       60.8 ± 9.70                                                                        62.5 ± 6.89                                                                        65.0 ± 4.08                                                                       --                                 Hb IV                                                                              61.0 ± 3.60                                                                       69.3 ± 3.58**                                                                      66.8 ± 5.60                                                                        67.7 ± 6.15                                                                        66.0 ± 0.00                                                                       --                                 6. URINARY OUTPUT [ml/30 min.]                                                PPF  2.60 ± 0.49                                                                       0.0 ± 0.00***                                                                      3.00 ± 0.61                                                                        3.10 ± 0.63                                                                        3.00 ± 0.53                                                                       --                                 Hb IV                                                                              5.16 ± 0.89b                                                                      6.58 ± 5.00a                                                                      12.66 ± 6.49*a                                                                     18.00 ±  8.69**a                                                                    8.67 ± 2.21**a                                                                    --                                 7. ARTERIAL BLOOD pH [UNITS]                                                  PPF  7.33 ± 0.04                                                                       7.34 ± 0.05                                                                        7.33 ± 0.03                                                                        7.33 ± 0.03                                                                        7.39 ± 0.13                                                                       --                                 Hb IV                                                                              7.31 ± 0.01                                                                       7.32 ± 0.01                                                                        7.29 ± 0.03                                                                        7.30 ± 0.02                                                                        7.32 ± 0.01                                                                       --                                 8. PaO.sub.2 [mm Hg]                                                          PPF  69.9 ± 8.29                                                                      70.08 ± 11.72                                                                       62.0 ± 4.67*                                                                       59.2 ± 8.09*                                                                       69.2 ± 5.88                                                                       --                                 Hb IV                                                                              65.8 ± 4.53                                                                       71.5 ± 6.69                                                                        74.4 ± 9.66c                                                                       65.6 ± 5.75                                                                        64.3 ± 2.15                                                                       --                                 9. PaCO.sub.2 [mm Hg]                                                         PPF  29.8 ± 3.24                                                                       31.2 ± 2.77                                                                        33.7 ± 2.98*                                                                       34.8 ± 2.11**                                                                      31.9 ± 5.15                                                                       --                                 Hb IV                                                                              28.7 ± 4.09                                                                       31.3 ± 4.89                                                                        31.2 ± 4.87                                                                        31.3 ± 4.44                                                                        29.5 ± 3.46                                                                       --                                 10. BLOOD P.sub.50 [mmHg]                                                     PPF  34.0 ± 0.61                                                                       33.7 ± 1.75                                                                        33.8 ± 3.01                                                                        34.0 ± 1.80                                                                        34.0 ± 1.80                                                                       --                                 Hb IV                                                                              32.9 ± 1.09                                                                       28.2 ± 2.05**b                                                                     29.2 ± 2.11**c                                                                     30.8 ± 1.46*c                                                                      31.3 ± 0.47                                                                       --                                 11. COLLOID OSMOTIC PRESSURE (COP) [mmHg]                                     PPF  18.2 ± 0.53                                                                       19.2 ± 0.06*                                                                       19.3 ± 0.77*                                                                       18.8 ± 0.60                                                                        18.3 ± 0.59                                                                       --                                 Hb IV                                                                              17.0 ± 0.24                                                                       19.2 ± 0.36***                                                                     19.0 ± 0.38***                                                                     18.5 ± 0.35***                                                                     17.8 ± 0.41*                                                                      --                                 12. PLASMA Hb [g/dl]                                                          PPF  0.00 ± 0.00                                                                       0.00 ± 0.00                                                                         0.00 ± 0.00                                                                       0.00 ± 0.00                                                                        0.00 ± 0.00                                                                       --                                 Hb IV                                                                              0.00 ± 0.00                                                                       3.73 ± 0.24***a                                                                    3.46 ± 0.31***a                                                                    3.03 ± 0.32***a                                                                    1.90 ± 0.22***a                        13. WBC [× 10.sup.9 ]                                                   PPF  6.27 ± 2.13                                                                       6.48 ± 1.16                                                                        7.25 ± 2.15                                                                        6.58 ± 1.53                                                                        6.55 ± 1.20                                                                       --                                 Hb IV                                                                              5.48 ± 0.87                                                                       3.88 ± 0.85*b                                                                      6.21 ± 2.50                                                                        9.00 ± 3.43                                                                        12.8 ± 4.92*                                                                       5.82 ± 1.92                    14. RBC [× 10.sup.12 ]                                                  PPF  5.10 ± 0.42                                                                       3.75 ± 0.42***                                                                     3.90 ± 0.27***                                                                     3.80 ± 0.39***                                                                     3.79 ± 0.32***                                                                    --                                 Hb IV                                                                              5.49 ± 0.30                                                                       4.12 ± 0.34***                                                                     4.06 ± 0.39***                                                                     4.15 ± 0.37***                                                                     3.95 ± 0.36***                                                                     3.89 ±  0.42**                 15. HEMOGLOBIN [g/dl]                                                         PPF  11.6 ± 0.82                                                                       8.4 ± 0.93***                                                                      8.8 ± 0.59***                                                                      8.6 ± 0.72***                                                                      8.7 ± 0.72***                                                                     --                                 Hb IV                                                                              12.4 ± 0.68                                                                       12.3 ± 0.54a                                                                       11.8 ± 0.68a                                                                       11.8 ± 0.84a                                                                       10.7 ± 0.90*b                                                                      9.5 ± 0.94***                  16. HEMATOCRIT [%]                                                            PPF  33.7 ± 2.85                                                                       24.6 ± 2.77***                                                                     25.5 ± 1.77***                                                                     24.9 ± 2.01***                                                                     24.9 ± 1.98***                                                                    --                                 Hb IV                                                                              37.2 ± 1.56c                                                                      27.9 ± 2.18***                                                                     27.6 ± 2.64***                                                                     28.1 ± 2.24***                                                                     26.9 ± 2.18***                                                                     29.0 ± 2.54***                 17. PLATELETS [× 10.sup.9 ]                                             PPF   388 ± 54.5                                                                        300 ± 45.6*                                                                        336 ± 51.8**                                                                       314 ± 39.8**                                                                       376 ± 36.2                                                                       --                                 Hb IV                                                                               453 ± 50.5                                                                        285 ± 52.1***                                                                      309 ± 42.5***                                                                      324 ± 49.7**                                                                       354 ± 23.6*                                                                        400 ± 175.5                   18. FIBRINOGEN [mg/dl]                                                        PPF   226 ± 31.8                                                                        165 ± 19.7**                                                                       180 ± 11.5**                                                                       191 ± 21.5*                                                                        334 ± 32.6***                                                                    --                                 Hb IV                                                                               217 ± 33.3                                                                        114 ± 29.8***b                                                                     117 ± 34.9**b                                                                      102 ± 29.6***b                                                                     391 ± 33.2***                                                                      500 ± 153.1**                 19. FIBRIN SPLIT PROD. [mcg/ml] [0 = <10 mcg/ml,                              1 = >10 < 40 mcg/ml, 2 = >40 mcg/ml]                                          PPF  0.00 ± 0.00                                                                       0.00 ± 0.00                                                                        0.16 ± 0.37                                                                        0.00 ± 0.00                                                                        0.00 ± 0.00                                                                       --                                 Hb IV                                                                              0.00 ± 0.00                                                                        1.67 ± 0.47***a                                                                   1.83 ± 0.37***a                                                                    1.83 ± 0.37***a                                                                    0.75 ± 0.43**a                                                                     0.00 ± 0.00                    20. PROTHROMBIN TIME [sec]                                                    PPF  10.8 ± 2.11                                                                       11.2 ± 1.64                                                                        9.25 ± 2.00                                                                        9.40 ± 1.00                                                                        8.00 ± 0.32*                                                                      --                                 Hb IV                                                                              11.0 ± 1.41                                                                       12.9 ± 1.92                                                                        15.5 ± 2.83*c                                                                      12.6 ± 2.29c                                                                       8.10 ± 0.10**                                                                      8.02 ± 0.04**                  21. TOTAL BILIRUBIN [mg/dl]                                                   PPF  0.33 ± 0.05                                                                       0.20 ± 0.08                                                                        0.23 ± 0.09                                                                        0.23 ± 0.09                                                                        0.27 ± 0.11                                                                       --                                 Hb IV                                                                              0.17 ± 0.16                                                                       0.28 ± 0.07                                                                        0.78 ± 0.54                                                                        0.38 ± 0.41                                                                        0.53 ± 0.55                                                                        0.45 ± 0.11*                   22. SGOT (AST) [Iu/L]                                                         PPF  39.6 ± 4.96                                                                       38.0 ± 0.43*                                                                       29.7 ±  5.85*                                                                      30.0 ± 3.66*                                                                       69.7 ± 7.49***                                                                    --                                 Hb IV                                                                              34.8 ± 9.19                                                                       41.0 ± 4.89b                                                                       52.0 ± 17.2c                                                                       62.0 ± 27.8b                                                                      380.0 ± 137.9***b                                                                   58.2 ± 21.2                    23. L D H [Iu/L]                                                              PPF  69.0 ± 12.0                                                                       50.8 ± 5.83**                                                                      60.0 ± 14.0                                                                        52.6 ± 9.63*                                                                        135 ± 35.5***                                                                    --                                 Hb IV                                                                              64.5 ± 15.1                                                                       43.3 ± 17.9                                                                        60.0 ± 40.8                                                                        86.0 ± 49.2                                                                       428.8 ± 336*                                                                         180 ± 122.5                   24. SGPT (ALT) [uI/L]                                                         PPF  46.2 ± 8.67                                                                       31.5 ± 7.99*                                                                       33.2 ± 8.82*                                                                       32.5 ± 8.48*                                                                       60.5 ± 5.44*                                                                      --                                 Hb IV                                                                              48.7 ± 12.2                                                                       37.2 ± 4.20                                                                        48.0 ± 17.2                                                                        60.0 ± 6.32a                                                                        191 ± 109.4*c                                                                     58.7 ± 16.7                     25. B U N [mg/dl]                                                            PPF  16.2 ± 2.13                                                                       16.2 ± 3.37                                                                        14.5 ± 2.43                                                                        14.2 ± 2.40                                                                        13.5 ± 2.08                                                                       --                                 Hb IV                                                                              15.2 ± 3.13                                                                       16.3 ± 3.25                                                                        15.8 ± 3.97                                                                        16.6 ± 4.53                                                                        20.4 ± 3.49*c                                                                      15.2 ± 1.46                    26. SERUM CREATININE [mg/dl]                                                  PPF  1.25 ± 0.18                                                                       1.23 ± 0.18                                                                        1.05 ± 0.10                                                                        1.07 ± 1.16                                                                        1.10 ± 0.12                                                                       --                                 Hb IV                                                                              1.42 ± 0.31                                                                       1.83 ± 0.62                                                                        1.70 ± 0.60c                                                                       1.36 ± 0.54                                                                        1.40 ± 0.37                                                                        1.26 ± 0.15                    27. SERUM SODIUM [mEq/l]                                                      PPF 137.0 ± 2.19                                                                      139.0 ± 2.86                                                                       140.5 ± 4.08                                                                       141.0 ± 4.31*                                                                      145.0 ± 1.25***                                                                    --                                 Hb IV                                                                             138.6 ± 3.39                                                                      135.0 ± 3.07                                                                       135.8 ±  2.03                                                                      138.2 ± 2.67                                                                       143.0 ± 4.00                                                                       143.0 ± 0.00                    28. SERUM POTASSIUM [mEq/l]                                                   PPF  3.45 ± 0.26                                                                       3.30 ± 0.30                                                                        3.50 ± 0.27                                                                        3.27 ± 0.28                                                                        4.35 ± 0.35***                                                                    --                                 Hb IV                                                                              3.32 ± 0.13                                                                       3.31 ± 0.17                                                                        3.55 ± 0.19                                                                        3.68 ± 0.30*                                                                       3.25 ± 0.05b                                                                       4.3 ± 0.00***                  29. SERUM CHLORIDE [mEq/l]                                                    PPF 101.2 ± 2.71                                                                      104.7 ± 2.50*                                                                      107.5 ± 3.56**                                                                     110.0 ± 2.53***                                                                    104.7 ± 2.75                                                                       --                                 Hb IV                                                                             104.2 ± 2.47                                                                      104.6 ± 4.34                                                                       105.0 ± 5.71                                                                       105.6 ± 3.85                                                                       106.5 ± 2.50                                                                       106.0 ± 0.00                    30. SERUM BICARB. [mEq/l]                                                     PPF  22.2 ± 2.64                                                                       22.7 ± 2.80                                                                        23.8 ± 1.60                                                                        24.3 ± 1.37                                                                        26.2 ± 4.92                                                                       --                                 Hb IV                                                                              23.2 ±  2.79                                                                      22.0 ± 2.08                                                                        22.8 ± 3.07                                                                        21.5 ± 3.20                                                                        22.0 ± 0.00                                                                        24.0 ± 0.00                    31. SERUM OSMOLALITY [mOsm/kg]                                                PPF 304.4 ± 11.3                                                                      299.5 ± 11.3                                                                       290.5 ± 8.65*                                                                      292.8 ± 9.91                                                                       293.7 ± 3.21*                                                                      --                                 Hb IV                                                                             290.5 ± 2.96                                                                      289.2 ± 5.15                                                                       288.2 ± 9.06                                                                       291.0 ± 6.22                                                                       293.5 ± 0.50                                                                       285.0 ± 0.00                    __________________________________________________________________________     M: ARITHMETIC MEAN                                                            ± SD: STANDARD DEVIATION                                                   DIFFERENCES BETWEEN THE GROUPS:                                               PPFHb I                                                                       PPFHb II                                                                      PPFHb III:                                                                    a  significant difference P < 0.001                                           b  significant difference P < 0.01                                            c  significant difference P < 0.05                                            DIFFERENCES BETWEEN T1 TO T2, T3, T4, T5:                                     ***  significant difference P < 0.001                                         **  significant difference P < 0.01                                           *  significant difference P < 0.05                                            DIFFERENCES BETWEEN THE GROUPS: PPFHb IV:                                     a  significant difference P < 0.001                                           b  significant difference P < 0.01                                            c  significant difference P < 0.05                                            n = 6 rabbits                                                            

                                      TABLE III                                   __________________________________________________________________________    Analysis of variance based on Difference in Scores                            4 groups analyzed                                                                               Treatment Times                                                               T1   T2   T3   T4    T5                                     Parameter         baseline                                                                           15 min.                                                                            1 hour                                                                             3 hours                                                                             24 hours                               __________________________________________________________________________    GENERAL SIGNS                                                                 Body Weight       --   --   --   NS    NS                                     Body Temperature  --   NS   NS   NS    NS                                     CARDIOVASCULAR FUNCTION                                                       Heart Rate        --   NS   NS   NS    NS                                     Systolic blood pressure                                                                         --   0.017                                                                              NS   NS    0.035                                                                   (P = 0.074)                                  Diastolic blood pressure                                                                        --   NS   NS   NS    NS                                     Arrhythmias       --   NS   NS   NS    --                                     Urinary output    --   <0.001                                                                             0.006                                                                              0.035 NS                                     (ml/30 min)                                                                   1 vs. 2                NS   0.013                                                                              NS    NS (P = 0.09)                          3 vs. 2                NS   NS   NS    NS                                     4 vs. 2                <0.001                                                                             <0.001                                                                             0.006 NS (P 0.09)                            RESPIRATORY FUNCTION                                                          Arterial Blood pH --   NS   NS   NS    NS (0.09)                              Pa O.sub.2        --   NS   0.035                                                                              0.006 NS (0.074)                             1 vs. 2                NS   NS   NS    NS                                     3 vs. 2                NS   0.006                                                                              0.014 NS (0.055)                             4 vs. 2                NS   NS   0.002 NS                                     Pa CO.sub.2       --   0.004                                                                              0.011                                                                              NS    NS                                     1 vs. 2                NS   NS   NS    NS                                     3 vs. 2                NS   NS   NS    NS                                     4 vs. 2                0.006                                                                              0.006                                                                              NS    NS                                     Blood P.sub.50    --   0.015                                                                              NS   NS    --                                     HEMATOLOGY                                                                    WBC               --   0.002                                                                              0.002                                                                              NS    NS                                     1 vs. 2                0.045                                                                              NS   NS    NS                                     3 vs. 2                NS   NS   NS    NS                                     4 vs. 2                <0.001                                                                             <0.001                                                                             NS    NS                                     RBC               --   NS   NS   NS    NS                                     Hemoglobin        --   <0.001                                                                             <0.001                                                                             0.003 NS                                     1 vs. 2                <0.001                                                                             <0.001                                                                             0.001 0.021                                  3 vs. 2                <0.001                                                                             0.001                                                                              0.004 NS                                     4 vs. 2                <0.001                                                                             <0.001                                                                             0.002 NS                                     Hemocrit          --   NS   NS   NS    NS                                     Platelets         --   0.030                                                                              0.037                                                                              0.010 NS                                     1 vs. 2                NS   NS   NS    NS                                     3 vs. 2                0.038                                                                              0.033                                                                              0.059 NS                                     4 vs. 2                0.008                                                                              0.006                                                                              0.001 NS                                     COAGULATION STUDIES                                                           Fibrinogen        --   NS   NS   NS    NS                                     Prothombin Time        NS   NS   NS    NS                                     LIVER FUNCTION                                                                SGOT              --   <0.001                                                                             NS   NS    0.043                                  1 vs. 2                <0.001                                                                             NS (0.052)                                                                         NS    NS                                     3 vs. 2                <0.001                                                                             NS   NS    NS                                     4 vs. 2                <0.001                                                                             NS   NS    0.016                                  LDH               --   NS   NS   NS (0.087)                                                                          NS (0.080)                             SGPT              --   NS   <0.001                                                                             NS    NS                                     1 vs. 2                NS   <0.001                                                                             NS    NS                                     3 vs. 2                NS   <0.001                                                                             NS    NS                                     4 vs. 2                NS   <0.001                                                                             NS    NS                                     RENAL FUNCTION                                                                BUN               --   NS   NS   NS    NS                                     Serum creatinine       NS   NS (0.095)                                                                         NS (0.069)                                                                          NS                                     Serum sodium           NS   NS   NS    NS                                     Serum Potassium   --   NS   NS   NS    NS                                     Serum chloride         NS   NS   NS    NS                                     Serum bicarbonate      NS   0.033                                                                              0.044 NS                                     1 vs. 2                NS   NS   NS    NS                                     3 vs. 2                NS   NS   0.009 NS                                     4 vs. 2                0.043                                                                              0.004                                                                              0.030 NS                                     Serum osmolarity  --   NS   NS   NS    NS                                     __________________________________________________________________________

                                      TABLE IV                                    __________________________________________________________________________    Students t-test For Paired Data                                               Comparison of 18 cases and 6 controls.                                                       Time 2      Time 3      Time 4      Time 5                                    5 min. baseline                                                                           1 hr-baseline                                                                             3 hr-baseline                                                                             24 hr-baseline                            mean ± se                                                                          (n) mean ± se                                                                          (n) mean ± se                                                                          (n) mean                                                                                  (n). se            __________________________________________________________________________    GENERAL DATA                                                                  Body weight                                                                   cases                                   0.11 ± 0.02                                                                       16   0.01                                                                                 18-. 0.02          control                                 0.09 ± 0.02                                                                       6    0.01                                                                                 6+-. 0.07                                                 NS          NS                         Body temperature                                                              cases          -0.22 ± 0.07                                                                       18  -0.14 ± 0.15                                                                       18  -0.19 ± 0.14                                                                       18    -13                                                                                 16-. 0.10          controls       -0.23 ± 0.13                                                                       6   -0.18 ± 0.15                                                                       6   -0.18 ± 0.09                                                                       6    0.00                                                                                 4+-. 0.00                         NS          NS          NS          NS                         Heart rate                                                                    cases          -18.7 ± 5.2                                                                        18  -15.9 ±  6.1                                                                       18   -6.8 ± 9.1                                                                        18   -6.4                                                                                 16-. 4.8           controls       -23.0 ± 9.2                                                                        5   -25.6 ± 10.8                                                                       5   -22.0 ± 11.7                                                                       5   -16.3                                                                                 4+-. 11.4                         NS          NS          NS          NS                         Systolic blood                                                                pressure                                                                      cases           +7.5 ± 3.2                                                                        18   +6.7 ± 3.0                                                                        18   +2.7 ± 3.3                                                                        18   +6.5                                                                                 13-. 2.9           controls        -4.2 ± 3.3                                                                        6    -5.0 ± 3.4                                                                        6    -3.3 ± 3.3                                                                        6    -4.0                                                                                 5+-. 2.9                          NS (P = 0.060)                                                                            0.050       NS          NS                         Diastolic blood pressure                                                      cases           10.0 ± 2.9                                                                        18    9.9 ± 2.8                                                                        18    5.0 ± 3.0                                                                        18    4.2                                                                                 13-. 2.6           controls         0.0 ± 2.9                                                                        6     0.0 ± 2.6                                                                        6     1.7 ± 3.3                                                                        6     2.0                                                                                 5+-. 4.1                          NS (P = 0.076)                                                                            NS (P = 0.063)                                                                            NS          NS                         Arrhythmia                                                                    cases             0 ± 0                                                                           18   0.18 ± 0.10                                                                       17   0.06 ± 0.06                                                                       17     0                                                                                  13-. 0             controls          0 ± 0                                                                           6      0 ± 6                                                                              0 ± 0                                                                       6          0 ± 0                                                                       4                                         NS          NS          NS          NS                         Urinary output                                                                (ml/30 min)                                                                   cases           -1.6 ± 0.6                                                                        18    3.7 ± 0.5                                                                        18    3.4 ± 0.8                                                                        18    1.5                                                                                 13-. 0.3           controls        -2.9 ± 0.2                                                                        4     0.1 ± 0.1                                                                        4     0.3 ± 0.8                                                                        4     0.1                                                                                 4+-. 0.1                          NS (P = 0.074)                                                                            <0.001      <0.001      0.003                      Respiratory Function Data                                                     Arterial Blood pH                                                             cases          -0.02 ± 0.02                                                                       18  -0.04 ± 0.03                                                                       18  -0.01 ± 0.03                                                                       18  -0.02                                                                                 15-. 0.07          controls        0.01 ± 0.01                                                                       6    0.00 ± 0.02                                                                       6    0.00 ± 0.02                                                                       6    0.08                                                                                 4+-. 0.06                         NS          NS          NS          0.030                      PaO.sub.2                                                                     cases            4.0 ± 3.5                                                                              8.3 ± 3.9                                                                        18   12.6 ± 4.8                                                                        18    3.9                                                                                 15-. 3.0           controls         0.9 ± 5.6                                                                             -8.0 ± 4.3                                                                        6   -10.8 ± 5.8                                                                        6    -0.8                                                                                 4+-. 3.6                          NS          0.035       0.017       NS                         PaCO.sub.2                                                                    cases            0.1 ± 1.3                                                                        18    1.6 ± 1.2                                                                        18    1.4 ± 1.2                                                                        18    1.7                                                                                 15-. 1.1           controls         1.4 ± 1.2                                                                        6     3.9 ± 1.2                                                                        6     5.0 ± 3.9                                                                        6     1.8                                                                                 4+-. 1.2                          NS          NS          NS          NS                         Blood P.sub.50                                                                cases           -4.4 ± 0.6                                                                        8    -2.8 ± 0.7                                                                        8    -1.4 ± 0.6                                                                        8   no data for                controls        -0.3 ± 0.6                                                                        3    -0.2 ± 1.3                                                                        3     0.0 ± 0.6                                                                        3   controls                                  0.004       NS          NS                                     COP                                                                           cases           0.10 ± 0.01                                                                       10   0.09 ± 0.01                                                                       10   0.07 ± 0.01                                                                       10   0.03                                                                                 8+-. 0.01          controls        0.05 ± 0.004                                                                      3    0.06 ± 0.01                                                                       3    0.03 ± 0.01                                                                       3    0.00                                                                                 3+-. 0.003                        0.007       NS          NS          0.030                      Hematology data                                                               WBC                                                                           cases           -1.4 ± 0.3                                                                        18   -0.6 ± 0.5                                                                        18    0.0 ± 0.6                                                                        18    3.6                                                                                 13-. 1.4           controls         0.1 ± 0.5                                                                        6     0.9 ± 0.5                                                                        6     0.2 ± 0.6                                                                        6     0.8                                                                                 4+-. 0.5                          0.036       NS          NS          NS (P = 0.094)             RBC                                                                           cases           -1.2 ± 0.1                                                                        6    -1.2 ± 0.1                                                                        18   -1.3 ± 0.1                                                                        18   -1.2                                                                                 13-. 0.2           controls        -1.4 ± 0.2                                                                        18   -1.2 ± 0.2                                                                        6    -1.3 ± 0.2                                                                        6    -1.1 ±                                                                            4.1                               NS          NS          NS          NS                         Hemoglobin                                                                    cases           -0.2 ± 0.2                                                                        18   -0.3 ± 0.2                                                                        18   -0.8 ± 0.3                                                                        18   -1.2                                                                                 13-. 0.4           controls        -3.2 ± 0.5                                                                        6    -2.8 ± 0.4                                                                        6    -3.1 ± 0.4                                                                        6    -2.6                                                                                 4+-. 0.4                          <0.001      <0.001      <0.001      NS (P = 0.099)             Hematocrit                                                                    cases           -8.0 ± 0.7                                                                        18   -7.8 ± 0.7                                                                        18   -8.2 ± 0.8                                                                        18   -7.6                                                                                 13-. 1.3           controls        -9.1 ± 1.4                                                                        6    -8.2 ± 1.1                                                                        6    -8.8 ± 1.3                                                                        6    -7.4                                                                                 4+-. 0.9                          NS          NS          NS          N                          Platelets                                                                     cases           -161 ± 18                                                                         18   -152 ± 16                                                                         18   -143 ± 14                                                                         18    -36                                                                                 13-. 41            controls         -88 ± 14                                                                         6     -53 ±  34                                                                        6     -74 ± 25                                                                         6     -12                                                                                 4+-. 30                           0.035       0.008       0.021       NS                         COAGULATION STUDIES                                                           Fibrinogen                                                                    cases             -89 ± 10                                                                        18    -102 ± 10                                                                        18    -108 ± 13                                                                        17     96                                                                                 13-. 36            controls          -61 ± 19                                                                        6      -46 ± 28                                                                        6      -52 ± 11                                                                        5      91                                                                                 4+-. 21                           NS          0.029       0.037       NS                         Prothrombin time                                                              cases            0.5 ± 0.6                                                                        16    1.0 ± 0.6                                                                        16    0.7 ± 0.4                                                                        16   -1.1                                                                                 13-. 0.6           controls         0.5 ± 0.9                                                                        6    -1.5 ± 0.4                                                                        6    -0.7 ± 1.0                                                                        5    -2.4                                                                                 4+-. 0.7                          NS          0.024       NS          NS                         LIVER FUNCTION                                                                Total B.sub.----                                                                             no data                                                        SGOT                                                                          cases           64.2 ± 6.1                                                                             50.7 ± 27.8                                                                       13   61.3 ± 23.3                                                                       15   236.9                                                                                13-. 80.0          controls        -9.4 ± 3.7                                                                            -10.2 ± 7.6                                                                        5    -8.2 ± 5.6                                                                        5    21.7                                                                                 3+-. 20.7                         <0.001      0.050       0.011       0.021                      LDH (.sub.--)                                                                 cases           0.34 ± 0.26                                                                       17   0.44 ± 0.29                                                                       17   0.64 ± 0.27                                                                       17   0.85                                                                                 15-. 0.41          controls       -0.28 ± 0.21                                                                       6   -0.16 ± 0.21                                                                       6   -0.04 ± 0.21                                                                       6    0.54                                                                                 4+-. 0.32                         NS          NS          NS          NS                         SGPT                                                                          cases           -0.9 ± 51.8                                                                       10   48.1 ± 4.4                                                                        9    13.2 ± 39.6                                                                       13   64.6                                                                                 14-. 35.8          controls        -8.5 ± 7.3                                                                        6    -6.8 ± 6.4                                                                        6    -7.5 ± 65                                                                         6    22.5                                                                                 4+-. 16.4                         NS          <0.001      NS          NS                         RENAL FUNCTION                                                                BUN                                                                           cases            0.4 ± 0.4                                                                        18    0.2 ± 0.4                                                                        18   -0.6 ± 0.8                                                                        18    1.8                                                                                 13-. 1.6           controls         0.0 ± 0.7                                                                        6    -1.7 ± 0.3                                                                        6    -2.0 ± 0.4                                                                        6    -2.0                                                                                 4+-. 0.8                          NS          0.016       NS          NS                         Serum creatinine                                                              cases           0.65 ± 0.33                                                                       18   0.84 ± 0.54                                                                       17   1.01 ± 0.47                                                                       17   0.56                                                                                 14-. 0.28          controls       -0.02 ± 0.03                                                                       6   -0.20 ± 0.05                                                                       6   -0.18 ± 0.07                                                                       6   -0.13                                                                                 4+-. 0.05                         NS (P = 0.059)                                                                            NS (P = 0.076)                                                                            0.023       0.027                      Serum sodium                                                                  cases           -2.7 ± 1.1                                                                        18   -0.8 ± 1.8                                                                        18   -0.6 ± 1.5                                                                        18    5.4                                                                                 14-. 1.4           controls         2.7 ± 0.5                                                                        6     3.8 ± 1.5                                                                        6     4.5 ± 1.9                                                                        6     7.5                                                                                 4+-. 0.6                          <0.001      NS          NS (P = 0.089)                                                                            NS                         Serum potassium                                                               cases          -0.14 ± 0.11                                                                       18  -0.03 ± 0.28                                                                       18  -0.23 ± 0.15                                                                       18  0.14                                                                                  14-. 0.23          controls       -0.15 ± 0.04                                                                       6   0.05 ± 0.03                                                                        6   -0.18 ± 0.14                                                                       6   1.03                                                                                  4+-. 0.18                         NS          NS          NS          NS (P = 0.067)             Serum chloride                                                                cases            0.0 ± 1.3                                                                        18    0.8 ± 0.9                                                                        18    1.2 ± 2.0                                                                        18    2.4                                                                                 14-. 1.6           controls         3.5 ± 0.9                                                                        6     6.3 ± 1.5                                                                        6     8.8 ± 0.9                                                                        6     2.5                                                                                 4+-. 1.9                          NS          0.007       0.002       NS                         Serum bicarbonate                                                             cases           -1.7 ± 0.7                                                                        18   -2.0 ± 0.6                                                                        18   -2.4 ± 0.8                                                                        18   -1.4                                                                                 14-. 0.9           controls         0.5 ± 0.3                                                                        6     1.7 ± 1.2                                                                        6     2.2 ± 0.9                                                                        6     4.3                                                                                 4+-. 1.9                          0.007       0.010       0.004       0.010                      Serum osmolarity                                                              cases          -10.1 ± 4.2                                                                        17  -11.4 ± 5.0                                                                        16  -14.5 ± 4.1                                                                        17  -12.7                                                                                 14-. 44            controls        -1.5 ± 2.9                                                                        4   -10.5 ± 6.7                                                                        4    -7.5 ± 10.6                                                                       4   -19.0                                                                                 2+-. 6.0                          NS          NS          NS          NS                         __________________________________________________________________________    PATHOLOGY DATA                                                                          mean score                                                                          n      P value                                                __________________________________________________________________________    Heart                                                                         cases     1.44  18     NS                                                     controls  1.00  6                                                             Lungs                          ≧1,<2                                                                           ≧2,<3                                                                      ≧3,<4                                                                          ≧4                                                                        Tot                    cases     1.89  18     NS (P = 0.067)                                                                        3(17)    14(77)                                                                            1(6)    0  18                     controls  1.58  6              4(67)     2(33)                                                                            0(0)    0   6                     Liver                                                                         cases     1.75  18     NS                                                     controls  1.92  6                                                             Kidneys                        ≧1,<2                                                                           ≧2,<3                                                                      ≧3,<4                                                                          ≧4                                                                        Tot                    cases     2.33  18     0.002   2(11)    11(62)                                                                             4(22)  1(6)                                                                             18                     controls  1.33  6              5(83)     1(17)                                                                            0       0   6                     Spleen                                                                        cases     1.00  9      NS                                                     controls  1.13  4                                                             __________________________________________________________________________

DATA ANALYSIS RESULTS

The three batches of hemoglobin here studied were:

Hb-I=Group 1

Hb-II=Group 3

Hb-III=Group 4

The batches were characterized as follows:

    ______________________________________                                                      Hb-I    Hb-II   Hb-III                                          ______________________________________                                        1.    Hemogobin, g/dl                                                                             14.0      13.0  10.0                                      2.    Oxyhemoglobin 90.3      91.2  98.6                                      3.    Carboxyhemoglobin                                                                           1.6       0.9   1.7                                       4.    Methemoglobin 8.8       10.6  2.7                                       5.    Oxygen Vol. % 17.5      16.2  13.6                                      6.    pH            7.5       6.55  7.0                                       7.    Sodium, mEq/L 118.5     102.3 119.2                                     8.    Potassium, mEq/L                                                                            3.88      4.16  2.44                                      9.    Chloride, mEq/L                                                                             118.0     117.3 120.9                                     10.   Osmolarity,   244       236   242                                             mOsm/Kg                                                                 11.   Endotoxins, EU/ml                                                                           <0.01     <0.01 <0.01                                     12.   Molecular wt. 85%       80%   90%                                             between 68,000-                                                               500,000                                                                 13.   Phospholipid  clear     clear clear                                           analysis by TLC                                                               silica gel plate                                                              developed in                                                                  iodine vapor                                                            ______________________________________                                    

These batches were compared to human Plasma Protein Fraction(Plasma-Plex-Armour Pharmaeutical Company). (Group 2=Control Group)

(A) Mortality

None of the animals in the 4 groups (6 rabbits in each group) died bythe end of the 24-hour observation period.

(B) Clinical Signs

During the first 3 hours following the administration of hemoglobin, theonly clinical sign was hemoglobinuria. At 24 hours, all animals appearednormal; i.e., with normal degrees of activity and eating and drinkingnormally. By that time, the hemoglobinuria had subsided.

No change occurred in body weight and temperature.

Gross Pathology

None of the animals presented at necropsy with hemoglobin extravasation.All the organs appeared grossly normal, with the exception of the liver,which appeared congested in about half the animals.

Histopathology

Heart: Focal areas of myocardinal "contracture" were found in the PPFgroup, as well as in the hemoglobin groups. The severity of the changewas graded as 1+ in the PPF group, and respectively 1.5+, 1.7+ and 1.25+in the Groups 1, 3 and 4. The difference was not statisticallysignificant.

Lungs: Areas of interstitial edema, congestion and cellular infiltrationwere found in all groups, including the PPF group. The severity ofchanges was graded 1.4+ for the PPF group, and respectively 2+, 1.8+ and1.8+ for Groups 1, 3 and 4. The difference, again, was not statisticallysignificant.

Liver: Changes found in the liver were more uniform than those observedin the other organs. Centrolobular vacuolization was graded as 1.4+ inthe PPF group, and respectively 1.5+, 1.8+ and 2+ in Groups 1, 3 and 4.The difference was not significant.

Kidneys: No glomerular alteration and no acute tubular necrosis orblockage by pigment casts were found. Tubular epithelial vacuolizationwas uniformly found in the subcapsular area. Extension from here towardthe cortico-medullary junction was graded as 1.6+ for the PPF group, andrespectively 1.8+, 2.15+ and 2.7+ for Groups 1, 3 and 4. The differencebetween PPF and the hemoglobin groups was significant only for Group 3.

Discussion and Conclusion

Both the chemical and histopathologic changes observed in this studywere mild-to-moderate and theoretically reversible. The determination ofsuch reversibility is currently being investigated with the period ofobservation being extended from 24 hours to 1 week.

EXAMPLE V Exchange Transfusion of Rabbits

Utilizing the experimental protocol set out in detail in Example IV,three groups of rabbits were hemorrhage-transfused. One group of sixrabbits (Groups A) had one-third of the estimated blood volume replacedwith a hemoglobin solution containing 1-2 endotoxin units per ml. Onegroup of four rabbits (Group B) had one-third of the estimated bloodvolume replaced with 5% PPF. Another group of six rabbits (Group C) hadone-third of the estimated blood volume replaced with a hemoglobinsolution of the present invention, this solution characterized asfollows:

    ______________________________________                                                   Run 1  Run 2    Run 3    Average                                   ______________________________________                                        1.  Hemoglobin, g/dl                                                                           11.6;    11.6;  11.4;  11.5                                  2.  Oxy-hemoglobin,                                                                            90.3;    90.2;  90.1;  90.2                                      %                                                                         3.  Carboxy-Hb, %                                                                              0.1;     0.3;   0.5;   0.3                                   4.  Met-Hb, %    9.6;     9.5;   9.7;   9.6                                   5.  Oxygen Vol. %                                                                              14.7;    14.6;  14.4;  14.6                                  6.  pH, Units    7.140;   7.161; 7.168; 7.156                                 7.  PCO.sub.2, Torr                                                                            14.1;    10.4;  10.2;  11.6                                  8.  PO.sub.2, Torr                                                                             147.5;   147.2; 147.0  147.2                                 9.  P.sub.50, Torr                                                                             28.0;                  28.0                                  10. Colloid Osmotic                                                                            20.7;    21.0;  20.9   20.9                                      Pressure, Torr                                                            11. Sodium, mEq/L                                                                              114.6    113.9  115.1  114.5                                 12. Potassium, Eq/L                                                                            3.72;    3.63;  3.70;  3.68                                  13. Chloride, mEq/L                                                                            111.0;   208.4; 107.2; 108.9                                 14. Phosphorus, ng %                                                                           0.097    0.097  0.097  0.097                                 15. Endotoxins,  0.29     0.19   0.23   0.23                                      EU/ml                                                                     16. Phospholipids, by            absent                                           TLC                                                                       17. Polymerization, by column 85% above tetrameric form.                          chromatography                                                            ______________________________________                                    

Comparisons for each group of one-third transfused rabbits during thetwenty-four hour period following transfusion was made with regard toplatelet numbers, white blood cell numbers, serum fibrinogen levels,prothrombin levels, and serum creatinine levels. The collected dataappears in FIGS. 2-6 respectively. In each of FIGS. 2-6, the trianglesrepresent mean values (± standard error) of the Group A rabbits, thesquares represent the mean values (± standard error) for the Group Brabbits, and the circles represent the means value (± standard error)for the Group C rabbits.

A comparison of the data represented by FIGS. 2-6 demonstrates thecross-linked hemoglobin solution of this invention caused no moralityand no clinically important signs. Using an analysis of variance, at 24hours post infusion there were no significant differences among thegroups except for a slight increase in systolic pressure and anelevation of SGOT. The elevation of systolic blood pressure wasconsidered clinically unimportant because it occurred within theclinically acceptable range (+20 mm Hg). The SGOT elevation wasconsidered spurious because of colorimetric interference by plasmahemoglobin. Although the test groups exhibited transient hemoglobinuria,no significant increase in BUN or serum creatinine was detected. Similarhistopathologic changes were found in both the PPF and the cross-linkedhemoglobin solution of the present invention groups. These alterationswere considered to be nonspecific and appeared to be reversible innature.

EXAMPLE VI Exchange Transfusion in Dogs

Preliminary studies on exchange transfusions in dogs were performed onbeagles and mongrel hounds with total blood volume exchanges rangingbetween 25%-75%. Dogs one, two and three are beagles weighingapproximately ten kilograms each, and dogs number four, five and six aremongrel hounds weighing approximately 20 kilograms each.

Dog number one (FIG. 8) is a beagle whose initial hematocrit of 24%indicated that he was anemic prior to transfusion. The anemia is of anundetermined type. This dog underwent a 40% exchange transfusion.Initial response was characterized by a rapid rise in hematocrit so thatafter 1.5 hours following exchange transfusion the hematocrit wasmeasured at 28%. Subsequently, the hematocrit rose above 36% by thesecond post transfusion day and remained in the range of 40% (102 daysfollowing transfusion). There was also a sustained rise in thehemoglobin which initially represented both intracellular hemoglobin andfree plasma hemoglobin as a result of the test infusion.

Chem 20 profiles obtained in a serial fashion, in general, failed toreveal any significant abnormalities although there was an indication ofincreased liver enzyme levels during the first nine days followinginfusion. Interpretation of these results is difficult because ofinterference produced by free hemoglobin in solution with standardizedautomated means of measuring liver enzymes.

Boston dog number two (FIG. 9) is a beagle who underwent a 25% exchangetransfusion also without adverse clinical effects. The initialhematocrit was appropriately depressed after the exchange transfusionand rapidly rose to exceed the resting level of 37% after 8 hours. Theelevated hematocrit level was sustained through the 92nd posttransfusion day and is confirmed by appropriate parallel increases inthe RBC count indicating increased red cell production. Similar liverenzyme changes were noted in the Creatanine and appeared to be slightlyelevated above resting levels. The remaining Chem 20 values did notchange significantly.

Boston dog number three (FIG. 10) is a beagle who underwent a 33%exchange transfusion. A similar rise above resting hematocrit levels wasnoted after the first day and this was sustained through the 78th posttransfusion day.

The liver enzymes showed a slightly different picture in this animal.Although the LDH appeared to have a transient elevation during the firsttwo days, the LDH levels were normal thereafter. In contrast, the SGOTand SGPT values appeared to be moderately elevated over the restinglevels for weeks after the initial transfusion. Clinically the animalappeared to have no adverse effects. However, the serum creataninelevels were slightly elevated following the infusion.

Dog number four (FIG. 11) is a mongrel weighing approximately 17kilograms and was used as a control animal. A 33% hemorrhage was inducedand the extracted blood volume was replaced with an equal amount of 5%human albumin. This was followed by the hematocrit level returning tonormal and slightly exceeding the resting value for the ensuing 81 daysserved.

Dog number five (FIG. 12) is a mongrel hound weighing approximately 20kilograms who underwent a 75% exchange transfusion with a significantdecrease in hematocrit followed by an increase in hematocrit exceedingthe resting level on the 7th post transfusion day. The animal sustaineda hematocrit which exceeded the resting level for the ensuing 43 daysfollowing the transfusion.

The liver enzymes showed a transient rise in LDH and SGOT whichgradually returned to the normal range. There was a single observationof an increase in SGPT which may have been aberrant data.

Dog number six (FIG. 13) is a mongrel hound weighing approximately 20kilograms who underwent a 75% exchange transfusion with similar findingsas that found in dog number five.

All of the dogs tested appeared to be well clinically and experienced arapid return to normal behavioral activity immediately after the acuteeffects of the anaesthesia subsided. There do not appear to be long termadverse effects observed in any of the tested animals.

SUMMARY

In a preliminary study of acute toxicity and efficacy of thecross-linked hemoglobin solution of the invention, five test dogsreceived single exchange transfusions replacing 25-75% of calculatedblood volume and one control dog underwent a 33% exchange replacementwith 5% albumin.

There was no mortality or clinical morbidity in any animal acutely orduring an extended observation up to twelve weeks. All test dogsexhibited normal activity immediately after replacement and experienceda rapid return of RBC parameters to the normal range within two weeks.Test animal chemistry profiles remained within normal limits except fora transient elevation of liver enzymes, a finding also observed in thecontrol dog (Dog #4). Arterial blood gas results on samples taken duringand immediately after the exchange transfusions indicated that normalPO₂ values were maintained in all animals including the two dogs whichunderwent 75% exchanges.

Dogs #5 and #6 both received exchange transfusions of approximately 75%of the blood volume (calculated by body weight). Dog #5 underwent a 50%blood volume hemorrhage followed by immediate replacement with 5%albumin solution as a first step in the exchange transfusion. This wasfollowed by a rapid removal of another 50% of the blood volume at whichtime Dog #5 became dyspnic manifested by a sudden increase inrespiratory rate from 14 per minute to 38 per minute associated withagonal breathing pattern. Because of the obvious clinical distressexhibited by Dog #5 at this time an equal amount of the cross-linkedhemoglobin solution of the invention solution was rapidly infused torestore the blood volume to normal. During the infusion of thecross-linked hemoglobin solution of the invention the respiratory ratereturned to 14 associated with a cessation of the labored breathingpattern. Blood gas determinations prior to the first hemorrhage, afterthe first replacement and after the second replacement indicated thatthe PO₂ remained within the normal range.

Dog #6 also underwent a 75% exchange transfusion similar to Dog #5 butthis time replacing the first 50% hemorrhage with an equal volume of thecross-linked hemoglobin solution of the invention and the second 50%hemorrhage with an equal volume composed of the cross-linked hemoglobinsolution of the invention and 5% albumin mixed in equal parts. After thefirst hemorrhage and replacement, there were no signs of respiratorydistress and there were no signs of respiratory distress during or afterthe second hemorrhage and replacement.

The arterial blood gas determinations during and after the exchangetransfusion indicated a normal PO₂ was maintained throughout theexchange.

These data suggest that the cross-linked hemoglobin solution of theinvention has both a volume expansion and an oxygen transport function.The response to a single transfusion of the cross-linked hemoglobinsolution of the invention did not appear to be associated with abnormalclinical behavior or abnormal chemistry of hematologic parameters,although a transient rise in liver enzymes was seen in both the testdogs and the control.

EXAMPLE VII Immunogenicity Studies

The immunogenicity of the hemoglobin solution produced in accordancewith the protocol of Example I and having the properties ascharacterized above (Invention Hemoglobin solution) was tested inprimates, subjected to three hemorrhage-transfusions of 1/3 thecalculated blood volume.

Six Coebus monkeys, of 4 Kg body weight, were sedated with Ketamine, 15mg/Kg body weight intramuscularly, and restrained. Sterile cannulae wereinserted percutaneously into one femoral artery and vein. Blood wasremoved from the artery in an amount corresponding to 2% of body weightin kilograms (approximately 1/3 of blood volume). The InventionHemoglobin solution was infused through the vein over a period of 30minutes. Blood samples (2.5 ml) were taken (1) before the removal ofblood, (2) 1 hour after the infusion of Invention Hemoglobin, (3) dailyfor 1 week, (4) weekly for 1 month, (5) monthly for three months. Thesera were tested for the presence of antibodies, using Ouchterlony'stest. The same experiment was carried out after 3 to 6 months. Thus,each animal underwent 3 Invention Hemoglobin infusions at three-monthintervals.

All animals survived the cycle of three hemorrhage-transfusions. No signof toxicity was noted (all animals appeared normal). The Ouchterlonytest resulted consistently negative for all sera, in all animals.

EXAMPLE VIII Material Persistance in the Circulation

This study was undertaken to demonstrate the unique vascular persistanceof the hemoglobin product of the invention. Since the early days ofhemoglobin research, it has been stated that hemoglobin is onlymaintained briefly in the circulation. The new technique and uniqueproduct not only works effectively but is maintained in the circulation.

To define the molecular weight of the product, we have developed datathat characterizes the unique hemoglobin-based temporary bloodsubstitute of this invention. We have measured the time elimination ofthe material in dog sera and have been able to characterize the bloodsubstitute as having the following characteristics by testing protocol.In the following examples, the hemoglobin used on the test animals isthe hemoglobin product produced in accordance with Example I and isreferred to as Invention Hemoglobin.

1. Determination of molecular weight distribution of hemoglobin in dogsera

Beagle dog sera which had been obtained during efficacy trials ofisovolemic exchange down to 5% hematocrit with Invention Hemoglobin werechecked concerning their hemoglobin-molecular weight distribution by gelpermeation chromatography (HP 1090 A). Changes in molecular weightdistribution with time after Invention Hemoglobin application were asfollows.

1.1 Analytical Conditions:

    ______________________________________                                        HPLC-Device:     HP 1090 A                                                    Integrator:      HP 3392                                                      Detector:        Diode array-UV-V/S/                                                           (Hewlett Packard)                                            GPC-column:      TSK G 3.000 SW 300 mm ×                                                 7.5 mm                                                       Eluent:          0.1 n K.sub.2 HPO.sub.4 (pH 7.0)                             Detection wavelengths:                                                                         260 nm (marker proteins) /                                                    405 nm (hemoglobin in dog                                                     sera)                                                        ______________________________________                                    

Results

These results are tabulated in the following Table V and thengraphically represented in FIG. 14. It is easily seen that vascularpersistance is maintained for much longer than the originally reported24-hour period demonstrated in other solutions.

                  TABLE V                                                         ______________________________________                                        Evaluation of Beagle Dog Sera                                                             Peak No.                                                                 % Hb   *1       *2       *3    *4                                      ______________________________________                                        Dog serum,        3.14     6.62   5.4                                          0 value                                                                       3 hr 20 min                                                                           5.8      129.11   184.45 239.78                                                                              719.35                                 24 h    5        170.11   188.34 230.87                                                                              461.74                                 48 h    4.5      511.98   236.29 249.42                                                                              249.42                                 96 h    3.8      910.38   165.00 172.50                                                                              127.50                                120 h    2.8      1,058.81 80.00  80.00 40.00                                 144 h    1.5      1,837.00 60.00  60.00 15.00                                 168 h    0.7      1,455.17 42.50  45.00 5.00                                  216 h    0.08     11.18    17.74  18.47 --                                    240 h    0.09     7.25     19.34  22.97 --                                    294      0.1      6.52     55.05  25.35 --                                    ______________________________________                                         *The numbers in each column represent the peak height in centimeters time     the full scale (in millivolts).                                          

Due to the elimination of hemoglobin the concentration decreasescontinuously (see Table V). Therefore, it had been necessary to workwith increasing amplifications ("full scale" (mV)) of the integratorsystem. In order to be able to compare the chromatographic peaks ofdifferent samples, peak areas or peak weights had to be recorded. Thiswas carried out as follows:

The peak heights were multiplied by the amplification "full scale". Theresulting values could be shown to be linear regarding hemoglobinconcentration; this has been verified by adding Invention Hemoglobin todog sera in hemoglobin concentrations from 1% to 7%. By plotting valueslog "peak height X full scale" vs. sampling time of sera (up to 294 h(Table V)), the kinetics shown in FIG. 14 can be obtained. Peak No. 1,representing the part of hemoglobin molecular weight distribution withthe highest molecular weight, shows the highest retention time value, asis expected from theory. Half-life values cannot be calculated from thiscurve because it does not reveal an ideal exponential function.

For the hemoglobin-components, represented by peaks 2, 3, and 4,respectively, the following intravascular half-life values were found:

    ______________________________________                                        peak 2: ca.84 h                                                               peak 3: ca.68 h                                                               peak 4: ca.24 h                                                               ______________________________________                                    

Decreasing half-life values, i.e., shorter intravascular retention timeswith decreasing molecular weight, confirm theoretical expectations.

Twenty-four hours after Invention Hemoglobin infusion, the molecularweight distribution in serum and in urine were examined. This comparisonshows that exclusively peak No. 4, standing for the 68,000-Hb-component,appears in the urine after that time.

The amount of the Hb-component (peak No. 4) in percent of the totalInvention Hemoglobin molecular weight distribution can be calculated byintegration of the peak areas (see the following Table VI).

                  TABLE VI                                                        ______________________________________                                                       Peak No. 4 Area                                                Sample         (% of Total Distribution)                                      ______________________________________                                        Invention Hemoglobin                                                                         48.0                                                           Serum 3 h 20 min                                                                             44.0                                                           24 h           38.9                                                           48 h           14.6                                                           ______________________________________                                    

The best representation of elimination from sera is demonstrated by thehalf-life evaluation curves in FIG. 14. In this figure, which is agraphical representation of the data of Table V, the data is arepresentation in percent of total Invention Hemoglobin remaining ineach subgroup molecular weight percentage.

EXAMPLE IX Sheep Experiments Objective

The objective of this study was to determine the efficacy of InventionHemoglobin solution infusion in splenectomized sheep through repeatedexchange transfusion and removal of red blood cells, and lowering ofhematocrit to approximately 5%. By this testing design, the potentialefficacy of Invention Hemoglobin solutions may be demonstrated bysurvivability in the absence of red blood content sufficient to sustainlife. The nature, degree, and duration of therapeutic effect was alsoassessed for each animal in this study.

Test Substance

Invention Hemoglobin solution as described above and as produced byExample I.

Test System

Purebred or mixed-breed sheep weighing 20.0 to 25.0 Kg were used forthis study. Animals were obtained from a commercial colony whose animalshave been certified for general health prior to the study and were knownto be sero-negative for Q-fever. The sheep were vaccinated for commonviral and bacterial pathogens; tested and treated for endoandectoparasites; and otherwise treated to minimize the effects of anydiseases which could create a variable. Animals were housed individuallyin pens bedded with shavings, fed guaranteed sheep ration and hadcontinuous access to potable water. Environmental parameters weremaintained at 70° F.±3° F., 45% RH± and 12 hr/12 hr light cycle. Theanimal room was operated as a conventional animal room, but technicianswore gloves, lab coats, and gowns when performing procedures.

Instrumentation

At least two weeks prior to study, sheep were fasted for 24 hours,pre-anesthetized with 0.2 mg I.M. atropine sulfate, anesthetized with 4%halothane by mask, intubated and maintained with approximately 2%halothane. Splenectomy was then performed using aseptic techniquethrough a ventral midline approach. The spleen was injected with 1:1000epinephrine during this procedure to mobilize any stored RBCs. Followingsplenectomy, bone marrow and liver biopsies were taken. Animals werethen allowed to recover for 14 days and monitored for continued goodhealth. Hematology values were compared before and after splenectomy todetermine any possible deleterious effects from the pre-study surgeryprocedure.

In order to conserve the test product Invention Hemoglobin, the initialexchanges were made with Ringer's lactate solution until a hematocrit ofapproximately 20% was achieved. Then additional exchanges were madeusing Invention Hemoglobin until the residual hematocrit was less than5%. For this study, 7 sheep which were screened for general good healthand pretreated for diseases were selected. Animals were uniquelyidentified for this study. In addition, 6 control sheep were tested in asimilar way with Hespan™ (hydroxyethyl starch solution) alone. Duringthis study, one sheep was randomly selected for testing on each of eighttesting days. The sheep were fasted for 24 hours prior to the study, andwater was withheld approximately 16 hours. Sheep were weighed,premedicated with 0.2 mg I.M. atropine sulfate and anesthetized with 4%halothane administered by facemask. When suitably anesthetized, thesheep were intubated and maintained on approximately 2% halothane.Anesthesia was titrated to Stage III, Plane 2-3 and maintained. Thefemoral artery was dissected using aseptic techniques and 15-gaugecatheters were placed in the vessel. Using similar techniques, thejugular was catheterized with a large bore Swan-Ganz catheter. Theanimal was then placed in a metabolic cage and was allowed to recoverfor two hours. Baseline blood pressure, measured by arterial cathetertransducer, and blood samples were obtained using the implantedcatheters. Twenty cc's of blood were removed each time for analysis ofhematology, chemistry, and blood gas determinations. Using anisovolumetric method, the blood volume was replaced in each animal, asfollows:

Procedure

1. Approximately 400 to 600 ml of the calculated circulatory volume (CV)was withdrawn from the femoral artery catheter. Blood pressurerecordings were taken again and after shock was produced (BP of 60/40)saline was infused with Ringer's lactate at 5 to 10 ml/minute to replacethe whole blood (equal volume). Blood pressure recordings and sampleswere again made following this infusion and the animal was allowed tostabilize.

2. After the stabilization period, blood pressures were recorded,followed by a second removal of another 400-600 ml of blood. Followingthis blood withdrawal, blood pressure values, samples, and clinicalevaluation for signs of shock were performed for approximately 10minutes.

3. After the 10 minute evaluation period, Invention Hemoglobin solutionwas administered at an infusion rate of 10 ml/minute. Blood pressure,samples, and clinical signs were monitored for 10 minutes to identifyconditions which would represent therapeutic effects.

4. Steps 2 and 3 were repeated until the hematocrit was reduced to below5%. Animals were observed continually and clinical signs were recorded.

5. Each animal was monitored so as to check for any signs associatedwith hemorrhage and treatment for a period of 2 hours. A terminal bloodsample of 20 cc's was taken for hematology, chemistry, and blood gasanalysis before removing the catheters and closing the catheterizationsites sterilely under light halothane anesthesia.

6. Changes in urinary output were monitored in a metabolic cage andrecorded through the hemorrhage treatment and recovery periods. Bloodgas values were also measured during each phase while arterial catheterwas in place.

7. When animals recovered from anesthesia, they were returned to theircages and given supportive therapy as necessary indicated by theirclinical condition. Comprehensive clinical monitoring and continuedsupportive therapy was provided for 21 days.

At the beginning of the study, baseline blood samples were drawn fromthe jugular vein and 24-hour urine samples were collected forurinalysis. At daily intervals and at the conclusion of the study,comparative blood samples were drawn, urine was collected and theophthalmological examinations were repeated. On Day 14, the sheep wereanesthetized as previously described and repeated liver and bone marrowbiopsies were performed. Animals that died were evaluated by grosspathological and histopathological evaluations. The spectrum of testsperformed were:

    ______________________________________                                                                   Blood Gases                                        Hematology  Clinical Chemistry                                                                           (arterial)                                         ______________________________________                                        RBC         SGOT           PO.sub.2                                           WBC         SGPT           PCO.sub.2                                          Platelet Count                                                                            LDG            pH                                                 Differential WBC                                                                          Alk Phos       O.sub.2 saturation                                 Hemoglobin  BUN            O.sub.2 content                                    Hemataocrit Creatinine                                                        REBC Morphology                                                                           Bilirubin (I and                                                  Reticulocyte Count                                                                        D)                                                                            Sodium                                                                        Potassium                                                                     Chloride                                                                      Calcium                                                                       Phosphorous                                                                   Total Protein                                                                 Albumin                                                                       Globulin                                                                      A/G/Ratio                                                                     Glucose                                                                       Cholesterol                                                                   Osmolarity                                                                    Carbon Dioxide                                                                Triglycerides                                                                 Iron                                                                          Iron Building                                                                 Capacity                                                                      Urinalysis                                                                    Specific Gravity                                                              pH                                                                            Protein                                                                       Glucose                                                                       Ketones                                                                       Hemoglobin                                                                    Hemoglobin Polymer                                                            Creatinine/Sedi-                                                              ment Exam                                                         Aqueous Humor                                                                 Hemoglobin                                                                    RBC         Serum and                                                                     Hemoglobin                                                                    Bovine Hemoglobin                                                             Antibody                                                          CSF         Polymerized                                                                   Hemoglobin                                                                    Hemoglobin     Ferritin                                           RBC         Heptiglobin                                                                                  Gross and Histo-                                   Coagulation Tests          logic Pathology                                    PT Lungs                   Heart                                              PTT Liver (biopsies        Kidneys (L and R)                                  included)                  Brain                                              Fibrinogen                 Eyes                                                                          Adrenals (L and R)                                                            Thyroid S                                                                     Bone Marrow                                                                   (biopsies                                                                     included)                                          Hemodynamics                                                                  Cardiac Output                                                                (thermal dilu-                                                                tion)                                                                         Arterial Pressure                                                             Central Venous                                                                Pressure                                                                      Pulmonary Artery                                                              Wedge Pressure                                                                ______________________________________                                    

All data from observations and tests were recorded on specific history,observation and testing report forms.

Statistical Analysis

Seven sheep were tested using the efficacy protocol. Each sheep hasbaseline blood samples and 24-hour urine samples performed. These datawere obtained at daily intervals during the follow-up period. For eachday of follow-up, statistical comparisons were performed using thepaired test comparing each follow-up measurement with the correspondingbaseline measurement. Of particular interest is a comparison of bloodand measurements immediately after the completion of all exchangetransfusions with the corresponding baseline measurements.

References

1. Blood Policy and Technology (Washington, D.C.: U.S. Congress, OfficeTechnology Assessment, OTA-H-260, January, pp. 133-150, 1985).

2. "Blood Groups and Blood Transfusion." In: The Merck VeterinaryManual. 5 ed. Merck Company, Inc. Rahway, N.J., pp. 42-49, 1979.

3. Kolata, R. J., Burrows, C. F. and Soma, L. R., "Shock:Pathophysiology and Management." In: Current Veterinary Therapy in SmallAnimal Practice. 7th ed., W. B. Saunders Company, Philadelphia, pp.32-48, 1980.

PRELIMINARY RESULTS OF THE SHEEP EFFICACY PROTOCOL are included in thisexample. The raw data regarding hemodynamic parameters is presented intabulated form in Tables VII (Test Animals) and VIII (Control Animals).The graphic presentation of the blood gas and hemodynamic data in FIGS.15-19 show that the test animals maintained arterial and venous oxygencontents well above the levels achieved in the controls which receivedno oxygen-carrying volume replacement during exchange transfusions. Alltest animals survived the exchange.

In contrast, all six of the control animals did not survive the acuteexchange transfusion. Six of the control animals showed deterioratingcardiac output as arterial and mixed venous oxygen contents, andhematocrits, fell below 10%.

In FIG. 18, the contribution of the Invention Hemoglobin as compared tothe sheep hematocrit is compared at decreasing hematocrit levels. FIG.18 demonstrates that at the end of the exchanges when the residualhematocrit is below 5% that approximately 80-90% of the arterial oxygencontent is contributed by the Invention Hemoglobin as compared toapproximately 10-20% which is contributed by both the liquid phase ofthe blood and the remaining red blood cells in the test animals.

Both the test group and the control sheep breathed room air at all timesduring the experiment. Both the test group and the control group showedan identical decline in arterial oxygen content during the firstexchange which lowered their respective groups from the baselinehematocrit down to the approximately 20% hematocrit levels. From thispoint on, the test group received Invention Hemoglobin in exchange forthe native sheep red blood cells. At this point, the figures demonstratethat the oxygen content is well maintained in the animals where bovinehemoglobin is used to replace the removed blood. In the case of thecontrols, there is decrease in the venous oxygen contents whichcontinues to parallel the decline in hematocrit since Hespan™ does notcarry a significant amount of oxygen except as a dissolved gas.

The association of oxygen delivery with the infused bovine hemoglobin isdemonstrated in FIG. 19 which clearly shows the departure of thedeclining oxygen delivery which is at first associated with thedeclining hematocrit in the initial exchange and which then increases inassociation with the increasing concentration of bovine hemoglobin whichis used to replace the sheep red blood cells during the exchange.

CONCLUSIONS

This study clearly demonstrates the efficacy of Invention Hemoglobin asan oxygen transport solution. The blood gas data and hemodynamic dataare consistent with the survivability of the test animals in contrast tothe control animals, none of which survived the acute exchangetransfusion. Since the residual hematocrits in all of the test animalswere less than 5% and, in some cases, between 1 and 2%, study clearlydemonstrates Invention Hemoglobin contributed significantly to adequateoxygen transport in the test animals. Furthermore, all of the testanimals survived on a long-term basis without intensive care orincreased inspired oxygen.

                                      TABLE VII                                   __________________________________________________________________________    Blood Gas Analysis - 7 Test Animals                                           EXCHANGE TRANSFUSION: OXYGEN MICE                                             ORIGINAL DATA                                                                 __________________________________________________________________________                                                    Ven     VEN                                   ART  VEN         OR      KOR CON                                                                              OR      HO2COM                SHEEP WTIKES HCT                                                                              POB  POB PCOB PH CONTENT BASELINE                                                                             CONTENT BASELINE              __________________________________________________________________________    GREEN 25.00  31.00                                                                            62.30                                                                              48.00                                                                             37.29                                                                              7.47                                                                             12.00   100.00 8.35    100.00                198   25.00  18.00                                                                            78.30                                                                              25.10                                                                             38.90                                                                              7.44                                                                             7.45    50.50  1.55    18.50                 Feb 24                                                                              85.10  17.00                                                                            54.50                                                                              19.30                                                                             44.10                                                                              7.33                                                                             7.55    58.98  2.00    33.50                 hemopure                                                                            25.00  16.00                                                                            65.70                                                                              80.70                                                                             25.20                                                                              7.54                                                                             7.85    56.00  1.00    16.77                 hemopure                                                                            85.00  11.00                                                                            57.89                                                                              14.30                                                                             34.40                                                                              7.43                                                                             7.90    61.70  2.10    85.15                 hemopure                                                                            25.00  4.50                                                                             70.90                                                                              17.00                                                                             38.00                                                                              7.52                                                                             8.30    64.84  3.95    47.21                 BLUE250                                                                             23.00  25.00                                                                            87.50                                                                              36.00                                                                             36.34                                                                              7.45                                                                             9.60    100.00 6.45    100.00                Mar 9 23.00  15.00                                                                            100.00                                                                             30.90                                                                             31.64                                                                              7.43                                                                             6.75    70.51  8.90    64.96                 hemopure                                                                            23.00  13.00                                                                            79.30                                                                              16.90                                                                             89.00                                                                              7.48                                                                             5.45    56.66  6.75    11.63                 hemopure                                                                            23.00  1.60                                                                             95.70                                                                              6.70                                                                              88.76                                                                              7.30                                                                             6.90    71.00  1.30    20.16                 BLUE250                                                                             83.00  20.00                                                                            95.20                                                                              45.20                                                                             38.90                                                                              7.50                                                                             9.95    100.00 7.85    100.00                May 12                                                                              23.00  16.00                                                                            95.30                                                                              38.60                                                                             38.60                                                                              7.44                                                                             9.70    97.49  8.90    40.00                 hemopure                                                                            23.00  3.00                                                                             86.30                                                                              19.60                                                                             27.20                                                                              7.50                                                                             7.85    78.06  3.50    40.20                 BLUE598                                                                             87.30  25.00                                                                            90.90                                                                              48.50                                                                             31.70                                                                              7.69                                                                             9.95    100.00 6.10    100.00                Aug 11                                                                              27.30  81.00                                                                            97.50                                                                              27.00                                                                             30.03                                                                              7.51                                                                             8.05    88.94  6.70    77.05                       87.30  13.00                                                                            85.70                                                                              31.40                                                                             36.30                                                                              7.45                                                                             6.30    63.32  1.05    30.33                 hemopure                                                                            87.30  10.00                                                                            62.60                                                                              24.34                                                                             34.30                                                                              7.40                                                                             7.60    76.30  8.30    37.70                 hemopure                                                                            87.30  4.50                                                                             70.00                                                                              19.70                                                                             38.90                                                                              7.46                                                                             7.89    78.36  8.50    40.90                 centblood                                                                           87.30  3.50                                                                             61.00                                                                              18.79                                                                             23.30                                                                              7.51                                                                             6.20    62.31  2.00    32.79                 centblood                                                                           87.30  8.30                                                                             83.00                                                                              13.90                                                                             38.40                                                                              7.46                                                                             6.09    60.30  1.75    20.69                 YELL699                                                                             90.00  20.00                                                                            87.00                                                                              47.00                                                                             26.90                                                                              7.44                                                                             11.25   100.00 9.30    100.00                Sept 10      19.00                                                                            90.00                                                                              30.30                                                                             25.00                                                                              7.43                                                                             7.25    64.64  4.55    48.98                 hemopure     12.00                                                                            96.00                                                                              89.00                                                                             25.00                                                                              7.29                                                                             6.85    60.09  8.45    26.34                 hemopure     7.00                                                                             86.00                                                                              25.10                                                                             36.50                                                                              7.37                                                                             6.65    59.11  2.70    29.03                 hemopure     8.09                                                                             95.34                                                                              26.30                                                                             29.94                                                                              7.43                                                                             7.25    64.44  3.40    36.54                 centblood    4.00                                                                             95.20                                                                              83.40                                                                             31.60                                                                              7.40                                                                             5.70    50.67  8.05    22.04                 centblood    8.00                                                                             95.30                                                                              16.00                                                                             38.70                                                                              7.41                                                                             2.60    24.89  1.25    13.44                 hemopure     1.50                                                                             95.30                                                                              20.70                                                                             23.00                                                                              7.43                                                                             3.90    24.67  1.75    19.00                 BLUE590                                                                             25.00  20.00                                                                            25.10                                                                              46.10                                                                             26.50                                                                              7.44                                                                             10.55   100.00 6.80    100.00                Sept 11      80.00                                                                            62.60                                                                              46.10                                                                             36.60                                                                              7.47                                                                             7.05    66.02  3.30    48.52                 hemopure     11.00                                                                            86.10                                                                              22.10                                                                             25.90                                                                              7.40                                                                             7.10    67.30  1.55    22.79                 hemopure     6.00                                                                             86.00                                                                              83.09                                                                             21.00                                                                              7.48                                                                             7.50    71.09  3.15    66.38                 hemopure     8.50                                                                             27.10                                                                              81.40                                                                             31.00                                                                              7.46                                                                             8.75    66.82  3.10    45.59                 BLUE209                                                                             34.50  20.00                                                                            92.60                                                                              47.00                                                                             38.00                                                                              7.60                                                                             18.65   100.00 9.05    100.00                Sept 14      18.00                                                                            89.00                                                                              36.10                                                                             33.00                                                                              7.44                                                                             8.19    64.3   3.50    43.90                 hemopure     8.00                                                                             147.90                                                                             89.00                                                                             15.60                                                                              7.59                                                                             8.90    64.57  8.10    38.34                 hemopure     3.56                                                                             82.00                                                                              83.00                                                                             27.50                                                                              7.40                                                                             9.95    70.75  4.05    60.35                 hemopure     3.00                                                                             81.50                                                                              83.64                                                                             77.00                                                                              9.00                                                                             9.45    77.00  9.73    71.43                 __________________________________________________________________________          PLASMA CARDIAC                                                                             nC.O.   OR HOW DEL     O2      O2 COM EXT                  SHEEP HGP    OUTPUT                                                                              BASELINE                                                                              DEL                                                                              BASELINE                                                                              AVOR                                                                              CONTURE BASELINE                                                                             FRACT                __________________________________________________________________________    GREEN DATA   6.38  100.00  30.30                                                                            10.00   4.48                                                                              11.25   100.00 31.90                198   NOT    6.10  97.15   17.61                                                                            55.00   5.90                                                                              11.32   124.00 36.00                Feb 24                                                                              TAKEN  6.15  65.60   18.50                                                                            3.73    4.75                                                                              7.00                                hemopure     3.50  55.26   10.15                                                                            31.37   5.05                                                                              8.19    72.00                       hemopure     4.69  74.1    14.02                                              hempoure                                                                            4.01   78.24 15.31   47.30                                                                            4.35    8.02                                                                              71.30   52.51                       BLUE250                                                                             DATA   5.61  100.00  23.42                                                                            100.00  3.15                                                                              7.60    100.00 28.81                Mar 9 NOT    4.40  115.51  19.00                                                                            61.81   3.05                                                                              10.05   141.20 57.03                hemopure                                                                            TAKEN  8.90  39.22   5.31                                                                             22.25   6.70                                                                              4.50    50.50  86.29                hemopure     3.04  27.75   9.78                                                                             41.50   9.60                                                                              7.00    102.72 81.16                BLUE250                                                                             DATE   4.40  100.00  19.05                                                                            100.00  2.70                                                                              5.17    100.00 27.11                May 12                                                                              NOT    4.22  95.91   17.81                                                                            93.69   6.00                                                                              18.48   241.29 70.05                hemopure                                                                            TAKEN  3.75  85.83   11.01                                                                            61.99   3.75                                                                              6.11    110.10 51.77                BLUE598                                                                             0.00   5.57  100.00  20.30                                                                            100.00  3.05                                                                              7.04    100.00 38.75                Aug 11                                                                              0.00   5.09  91.26   16.50                                                                            81.20   4.15                                                                              7.76    98.67  48.91                      0.00   6.06  100.00  14.00                                                                            60.97   4.45                                                                              9.08    125.70 70.57                hemopure                                                                            1.72   6.64  83.30   18.90                                                                            63.55   5.30                                                                              9.61    114.63 69.84                hemopure                                                                            5.22   3.10  55.64   8.10                                                                             44.30   4.70                                                                              5.34    67.94  65.28                centblood                                                                           3.04   3.44  61.96   7.81                                                                             30.47   4.00                                                                              5.29    67.30  67.73                centblood                                                                           4.00   6.03  86.91   10.68                                                                            52.38   4.25                                                                              7.52    95.67  70.81                YELL699                                                                             0.00   6.02  100.00  82.39                                                                            100.00  1.93                                                                              4.05    100.00 17.31                Sept 10                                                                             0.00   5.54  91.87   13.05                                                                            59.21   8.70                                                                              5.12    126.62 34.97                hemopure                                                                            8110.00                                                                              3.04  63.02   8.98                                                                             20.39   4.60                                                                              5.76    162.22 64.16                hemopure                                                                            2600.00                                                                              3.03  63.52   8.70                                                                             27.54   2.95                                                                              5.22    100.09 59.45                hemopure                                                                            4500.00                                                                              4.29  71.16   10.78                                                                            45.03   3.05                                                                              5.70    100.74 53.17                centblood                                                                           3600.00                                                                              3.91  64.04   7.69                                                                             22.09   3.63                                                                              4.98    121.68 63.98                centblood                                                                           2340.00                                                                              3.89  64.31   3.76                                                                             16.00   1.55                                                                              2.00    51.36  55.28                hemopure                                                                            4220.00                                                                              4.77  79.01   6.41                                                                             87.40   2.15                                                                              2.54    87.61  55.22                BLUE590                                                                             0.00   3.68  100.00  13.64                                                                            100.00  3.95                                                                              4.05    100.00 25.50                Sept 11                                                                             0.00   4.13  114.00                                                     hemopure                                                                            3000.00                                                                              8.08  77.00   7.15                                                                             52.68   5.58                                                                              5.59    115.26 78.10                hemopure                                                                            4960.00                                                                              4.14  114.30  11.00                                                                            61.30   4.35                                                                              6.43    128.50                      hemopure                                                                            6304.00                                                                              3.18  87.05                                                      BLUE209                                                                             0.00                                                                    Sept 14                                                                             0.00                                                                    hemopure                                                                      hemopure                                                                      hemopure                                                                            7652.00                                                                 __________________________________________________________________________

                                      TABLE VIII                                  __________________________________________________________________________    BLOOD GAS ANALYSIS - 6 Control Animals                                        __________________________________________________________________________                 ART VEN         O2     HO2 COM                                                                              O2     HO3    PLASMA                            PO2 PO2 PCO2                                                                              PH  CONTENT                                                                              BASELINE                                                                             CONTENT                                                                              BASELINE                                                                             HGD                  __________________________________________________________________________    55     22.30                                                                            16.00                                                                            89.60                                                                             34.80                                                                             36.50                                                                             7.49                                                                              6.30   100.00 2.70   100.00                      HESPAN    9.00                                                                             90.70                                                                             26.50                                                                             33.70                                                                             7.43                                                                              3.65   57.94  9.60   22.22                       CONTROL   7.50                                                                             96.40                                                                             23.40                                                                             32.20                                                                             7.46                                                                              3.15   50.00  9.60   14.81                                 6.50                                                                             121.60                                                                            17.20                                                                             27.10                                                                             7.43                                                                              1.90   30.16  0.20   7.41                                  4.00                                                                             129.00                                                                            10.10                                                                             19.30                                                                             7.41                                                                              1.65   26.19  0.20   7.41                        56     21.40                                                                            19.00                                                                            90.00                                                                             37.10                                                                             40.70                                                                             7.43                                                                              8.25   100.00 3.40   100.00                      HESPAN    14.00                                                                            87.70                                                                             31.20                                                                             36.90                                                                             7.46                                                                              5.85   70.91  2.00   50.02                       CONTROL   10.00                                                                            89.00                                                                             25.00                                                                             32.40                                                                             7.49                                                                              4.35   52.73  6.05   25.00                                 7.50                                                                             96.80                                                                             24.10                                                                             32.20                                                                             7.49                                                                              3.45   41.02  0.40   11.74                                 6.50                                                                             108.90                                                                            20.10                                                                             32.90                                                                             7.45                                                                              2.70   32.73  0.20   5.88                                  5.50                                                                             110.10                                                                            17.70                                                                             32.30                                                                             7.42                                                                              2.35   28.48  0.15   4.41                                  5.00                                                                             115.00                                                                            15.90                                                                             27.00                                                                             7.38                                                                              2.15   26.06  0.15   4.41                        57     20.00                                                                            23.00                                                                            91.10                                                                             29.40                                                                             29.20                                                                             7.44                                                                              9.60   100.00 5.40   100.00                      HESPAN    17.00                                                                            71.00                                                                             31.50                                                                             36.70                                                                             7.47                                                                              7.00   72.92  2.50   46.30                       CONTROL   13.00                                                                            99.00                                                                             31.70                                                                             20.00                                                                             7.51                                                                              5.80   60.42  2.00   37.04                                 9.00                                                                             91.00                                                                             23.60                                                                             34.00                                                                             7.45                                                                              3.95   41.15  0.85   15.74                                 7.00                                                                             101.20                                                                            22.20                                                                             32.20                                                                             7.47                                                                              2.75   28.65  0.40   7.41                                  6.00                                                                             106.30                                                                            17.90                                                                             30.70                                                                             7.48                                                                              2.50   26.04  0.25   4.63                                  5.00                                                                             115.70                                                                            18.00                                                                             25.20                                                                             7.48                                                                              2.25   23.44  0.20   3.70                        58     10.20                                                                            15.00                                                                            90.90                                                                             35.00                                                                             37.70                                                                             7.48                                                                              6.90   100.00 3.35   100.00                      HESPAN    12.00                                                                            89.60                                                                             29.00                                                                             35.80                                                                             7.47                                                                              5.45   81.00  1.05   55.22                       CONTROL   8.50                                                                             94.20                                                                             24.00                                                                             34.00                                                                             7.46                                                                              3.70   53.62  1.15   34.33                                 7.00                                                                             99.80                                                                             20.00                                                                             33.00                                                                             7.44                                                                              3.30   47.83  0.85   22.39                                 5.00                                                                             109.60                                                                            19.40                                                                             27.00                                                                             7.47                                                                              3.05   44.20  0.30   0.96                                  4.50                                                                             120.00                                                                            20.00                                                                             23.90                                                                             7.44                                                                              2.75   39.06  0.25   7.44                        89     19.00                                                                            17.00                                                                            86.00                                                                             34.90                                                                             39.60                                                                             7.44                                                                              7.85   100.00 3.40   100.00                      HESPAN    12.00                                                                            103.10                                                                            36.20                                                                             30.30                                                                             7.53                                                                              6.10   77.71  2.75   80.88                       CONTROL   10.00                                                                            96.00                                                                             92.50                                                                             33.30                                                                             7.47                                                                              4.45   56.69  1.30   38.24                                 7.00                                                                             88.90                                                                             24.40                                                                             34.80                                                                             7.45                                                                              3.20   40.74  0.90   26.47                                 5.50                                                                             99.90                                                                             18.30                                                                             34.80                                                                             7.45                                                                              2.55   32.48  0.40   11.74                       510    21.00                                                                            22.00                                                                            75.60                                                                             43.20                                                                             36.30                                                                             7.49                                                                              8.90   100.00 4.75   100.00                      HESPAN    20.00                                                                            74.90                                                                             37.40                                                                             31.00                                                                             7.49                                                                              7.95   89.33  4.30   90.53                       CONTROL   11.00                                                                            101.50                                                                            31.60                                                                             29.60                                                                             7.53                                                                              5.35   60.11  2.10   44.21                                 8.50                                                                             100.00                                                                            24.20                                                                             25.50                                                                             7.49                                                                              3.90   43.82  0.90   18.95                                 7.00                                                                             103.60                                                                            18.90                                                                             27.00                                                                             7.49                                                                              3.65   41.01  0.65   13.68                                 5.00                                                                             116.50                                                                            17.00                                                                             21.10                                                                             7.45                                                                              2.85   32.02  0.80   16.84                       __________________________________________________________________________            CARDIAC                                                                              nC.C.   C2  HO2 DEL       O2              EXT                          OUTPUT BASELINE                                                                              DEL BASELINE                                                                              AVCOR CONTURE BASELINE                                                                              FRACT                __________________________________________________________________________    55      5.62   100.00  15.90                                                                             100.00  2.00  9.10    100.00  57.80                HESPAN  7.08   125.98  11.60                                                                             72.96   3.06  9.70    106.59  83.60                CONTROL 6.51   115.64  9.20                                                                              57.86   2.75  8.00    87.91                                4.74   84.34   4.60                                                                              25.16   1.70  3.60                                         3.01   53.50   2.20                                                                              13.84   1.45  2.00    21.98   90.90                56      5.77   100.00  22.24                                                                             100.00  4.05  13.00   100.00  58.00                HESPAN  5.78   100.17  15.00                                                                             71.04   3.05  10.40   79.51   65.80                CONTROL 6.06   105.03  12.32                                                                             25.60   3.50  9.91    75.76   80.40                        6.02   104.32  9.70                                                                              43.62   3.05  0.50    65.60   88.50                        5.45   94.45   4.00                                                                              30.94   2.50  6.37    48.70   92.60                        4.82   83.54   5.29                                                                              23.79   2.20  4.96    37.92   93.80                        2.76   47.83   2.77                                                                              12.46   2.00  2.50    19.78   93.10                57      4.50   100.00  21.60                                                                             100.00  4.20  9.45    100.00  43.00                HESPAN  4.83   107.33  16.90                                                                             78.24   4.50  10.07   115.03  64.60                CONTROL 5.59   124.22  16.20                                                                             75.00   3.00  10.62   112.30  65.60                        6.19   137.54  12.20                                                                             56.48   3.10  9.59    101.48  79.60                        6.91   153.54  9.50                                                                              43.90   5.25  0.18    85.93   85.50                        5.98   131.54  7.60                                                                              34.26   2.25  6.66    70.48   90.00                        6.01   151.33  7.66                                                                              35.46   2.05  6.98    73.04   91.10                58      5.71   100.00  21.60                                                                             100.00  3.85  11.10   100.00  51.60                HESPAN  4.07   106.30  10.04                                                                             87.22   3.80  12.67   14.14   47.30                CONTROL 7.31   128.02  14.86                                                                             68.80   2.55  10.24   92.25   68.90                        7.64   133.00  13.95                                                                             64.18   2.53  10.70   96.40   77.30                        4.92   121.02  11.58                                                                             53.61   2.75  10.44   94.05   90.20                        4.18   73.20   6.38                                                                              29.26   2.50  3.74    51.71   90.90                89      5.17   100.00  21.36                                                                             100.00  4.45  12.11   100.00  56.69                HESPAN  7.04   134.17  22.60                                                                             105.01  3.35  12.41   94.12   54.92                CONTROL 5.07   113.54  13.75                                                                             64.37   3.15  9.783   93.27   70.78                        6.51   125.92  10.96                                                                             51.31   3.01  10.31   87.39   94.10                        5.04   112.96  7.04                                                                              36.70   2.15  6.61    70.43   94.29                510     5.54   100.00  22.62                                                                             100.00  4.15  10.55   100.00  44.62                HESPAN  5.93   107.04  21.63                                                                             95.63   3.65  9.93    94.12   45.90                CONTROL 6.60   119.13  16.20                                                                             71.62   3.25  9.84    93.27   60.74                        6.70   120.94  11.99                                                                             53.01   3.00  9.22    87.39   76.90                        5.40   97.47   9.04                                                                              39.96   3.00  7.43    70.43   82.20                        6.92   124.91  9.05                                                                              40.01   2.05  6.51    61.71   71.96                __________________________________________________________________________

EXAMPLE X Dog Experiments Objective

Invention Hemoglobin was tested for efficacy, tolerability, and sideeffects on beagle dogs. The experimental arrangement was intended toanswer the question of whether the preparation studied was able toassume the oxygen transport function and the volume replacement in caseof severe losses of endogenous blood, and how rapidly the hemoglobinsupplied is excreted from the body. The tolerability and the occurrenceof unexpected side effects was also determined secondarily.

There is an indication for an O₂ transportation solution only when theremaining amount of endogenous hemoglobin is no longer able to satisfythe O₂ requirement of the tissue. From this it follows that the anemiaof the experimental animals must be so severe that at least detectableinjuries occur from the O₂ deficiency, and at the same time, theadministration of the hemoglobin solution demonstrably prevents theseinjuries.

Test Substance

Invention Hemoglobin solution as described above and as produced by theprocess of Example I.

Experimental Model

7 purebred beagle dogs were used for study who had been splenectomizedapproximately 3 weeks before the beginning of the study.

Instrumentation

After premedication and the initiation of anesthesia, an isovolemichemodilution was carried out through peristaltic pumps in a controlledmanner on 4 animals with Invention Hemoglobin solution up to a residualhematocrit of 5%, and on 3 control animals with a hydroxyethyl starchsolution (HES) whose ionic composition and colloid osmotic pressure wereequivalent to those of the Invention Hemoglobin solution. A number ofparameters were determined during the exchange transfusion and thefollow-up period of 10 days.

Dog Test Results

In this study, 4 test dogs received exchange transfusion with InventionHemoglobin under general anesthesia over a 3-hour period withmeasurements approximately every 10 minutes. All test dogs survived theacute exchange transfusion with residual hematocrits below 5%. Thehemodynamic and blood gas analysis revealed that all test dogs were welloxygenated at the end of the procedure in contrast to the 3 control dogswhich did not survive the exchange transfusion with the hydroxyethylstarch solution. The 3 control dogs had evidence of inadequateoxygenation associated with the decreasing hematocrit levels. FIGS.20-24 demonstrate the differences in the test and control groups forselected parameters measured.

Conclusion

The preliminary results of this efficacy study demonstrate thatInvention Hemoglobin solution contributes to normal oxygenation of testdogs which are severely depleted of red blood cell mass. In contrast tothe control dogs which could not survive with very low residualhematocrit levels, the test animals all survived the acute exchangetransfusion. The conclusion of this study is that it is evident thatInvention Hemoglobin solution transports oxygen under extreme conditionsof severe red blood cell loss.

Having now fully described the invention, it will be readily apparent toone skilled in the art that many changes and modifications may be madethereto without departing from the spirit or scope thereof.

We claim:
 1. A blood-substitute comprising of from about 5 to about 25grams of crosslinked hemoglobin per deciliter of an aqueouspharmaceutically acceptable carrier medium, said blood-substitute beingsubstantially free of cell stroma, non-hemoglobin proteins and pyrogensand said blood-substitute being sufficiently purified with respect toendotoxin so that the blood-substitute does not, upon in vivoadministration to a mammal in a significant dosage, cause complementactivation.
 2. A blood-substitute of claim 1 having an endotoxin levelof less than about 0.02 endotoxin units per milliliter (EU/ml).
 3. Ablood-substitute of claim 1 wherein said hemoglobin comprises humanhemoglobin.
 4. A blood-substitute of claim 2 wherein said hemoglobincomprises bovine hemoglobin.
 5. A blood-substitute of claim 3 whereinsaid mammal comprises a human being.
 6. A blood-substitute of claim 4wherein said mammal comprises a human being.
 7. A blood-substitutecomprising an aqueous solution of crosslinked hemoglobin, saidblood-substitute characterized by:a) an endotoxin concentration of lessthan about 0.02 endotoxin units per milliliter (EU/ml) determined by achromogenic Limulus Amebocytic Lysate (LAL) assay against an end-pointreaction made by a series of dilutions of a reference endotoxin solutionand thereafter interpolating from a standard regression curve made fromcolorometric readings from said dilutions; b) a phospholipidconcentration of less than about 1 nanogram per milliliter; c) ahemoglobin molecular weight distribution of greater than about 90% inthe range of 68,000-500,000 daltons; d) an osmolarity as measured byfreezing point depression in the range of 180-320 milliosmoles perliter; e) a hemoglobin content of from about 5 to about 25 grams perdeciliter; f) a methemoglobin content of less than about 20%, by weight;g) a P₅₀ in the range of about 18-36 mm Hg; and h) being substantiallyfree of red blood cell stroma, non-hemoglobin proteins and pyrogens. 8.A blood-substitute of claim 7 wherein said hemoglobin comprises humanhemoglobin.
 9. A blood-substitute of claim 7 wherein said hemoglobincomprises bovine hemoglobin.
 10. A mammalian blood-substitute comprisingcrosslinked hemoglobin suspended in an aqueous medium, saidblood-substitute having: crosslinked hemoglobin having a molecularweight distribution of greater than about 90% in the range of about68,000 to 500,000 daltons; an osmolarity as measured by freezing pointdepression in the range of about 180-320 milliosmoles per liter ofsolution; a final hemoglobin content of from about 9 to about 13 gramsper deciliter; a methemoglobin content of less than about 10%, byweight; physiologic levels of sodium chloride and potassium chloride;less than about one nanomole of phospholipid per milliliter; less thanabout one part per million of crosslinking agent; a P₅₀ in the range ofabout 18-36 mm Hg; and an endotoxin level of less than about 0.5endotoxin units per milliliter (EU/ml).
 11. A mammalian blood-substituteof claim 10 wherein said hemoglobin comprises human hemoglobin.
 12. Amammalian blood-substitute of claim 10 wherein said hemoglobin comprisesbovine hemoglobin.
 13. A mammalian blood-substitute of claim 11 whereinsaid endotoxin level is less than about 0.02 EU/ml.
 14. A mammalianblood-substitute of claim 12 wherein said endotoxin level is less thanabout 0.02 EU/ml.
 15. A mammalian blood-substitute of claim 10 furthercharacterized by the fact that, in vivo, in a therapeuticallyefficacious dosage, it:a) does not activate complement; b) does notcause hemorrhagic disorders; c) does not cause abnormal plateletfunction or aggregation; d) does not cause abnormal prothrombin times(PT); e) does not cause abnormal partial thromboplastin times; f) doesnot interfere with blood typing or cross-matching; g) is non-toxic tothe kidneys of normal healthy mammals at concentrations of 3.5 grams perkilogram body weight and 8 grams per deciliter circulating blood volume;h) exhibits circulating persistence of at least seven days; and i) actsas a stimulus to accelerated erythropoiesis.
 16. A blood-substitutecomprising intramolecularly and/or intermolecularly crosslinkedhemoglobin in an aqueous pharmaceutically acceptable carrier medium,said hemoglobin being crosslinked and said blood-substitute beingsubstantially free of cell stroma, non-hemoglobin proteins and pyrogensand said blood-substitute, upon in vivo administration to a mammal in atherapeutically significant dosage, having an endotoxin level which doesnot cause complement activation.
 17. A blood-substitute of claim 16having an endotoxin level of less than about 0.5 EU/ml.
 18. Ablood-substitute of claim 16 having an endotoxin level of less thanabout 0.02 EU/ml.
 19. A blood-substitute of claim 18 wherein saidhemoglobin comprises human hemoglobin.
 20. A blood-substitute of claim18 wherein said hemoglobin comprises bovine hemoglobin.
 21. Ablood-substitute of claim 19 wherein said mammal comprises a humanbeing.
 22. A blood-substitute of claim 20 wherein said mammal comprisesa human being.
 23. A blood-substitute comprising an aqueous solution ofcrosslinked hemoglobin, said blood-substitute:a) having a P₅₀ measuredunder human physiologic conditions in the range of from about 18 toabout 36 mm Hg; b) being substantially free of red blood cell stroma andnon-hemoglobin proteins; and c) having an endotoxin level of less thanabout 0.5 endotoxin units per milliliter (EU/ml).
 24. A blood-substituteof claim 23 having an endotoxin level of less than about 0.02 EU/ml. 25.A blood-substitute of claim 24 wherein said hemoglobin comprises humanhemoglobin.
 26. A blood-substitute of claim 24 wherein said hemoglobincomprises bovine hemoglobin.
 27. A hemoglobin solution for transportingand releasing oxygen to living cells comprising hemoglobin in an aqueouspharmaceutically-acceptable carrier medium, said hemoglobin solutionbeing substantially free of red blood cell stroma and having anendotoxin level of less than about 0.5 endotoxin units per milliliter(EU/ml).
 28. A hemoglobin solution of claim 27 wherein said hemoglobinis crosslinked.
 29. A method for producing a hemoglobin solutionsubstantially free of endotoxins by performing the following sequence ofsteps under substantially endotoxin free conditions:a) disrupting thered blood cell fraction of blood without prior washing to produce ahemoglobin-containing solution; b) clarifying said hemoglobin-containingsolution to produce a substantially stromal-free hemoglobin-containingsolution; c) microporously filtering said substantially stromal-freehemoglobin-containing solution to produce a partially sterilized,stromal-free, hemoglobin-containing solution; d) ultrafiltering saidpartially sterilized, stromal-free hemoglobin-containing solution toproduce a size-separated, partially sterilized, stromal-free,hemoglobin-containing solution; and e) chromatographically treating saidsize-separated, partially-sterilized, stromal-free,hemoglobin-containing solution in a high performance liquidchromatographic column containing a packing for the ion-exchangeaffinity separation of endotoxin from hemoglobin under conditionssufficient to produce a substantially phospholipid-free, substantiallyendotoxin-free hemoglobin solution.
 30. A method of claim 29 includingthe additional steps of crosslinking hemoglobin in said substantiallyphospholipid-free, substantially endotoxin-free hemoglobin solution toproduce a semi-synthetic blood-substitute.
 31. In the preparation of anultra pure hemoglobin solution, from mammalian blood with an endotoxinlevel of less than 7 endotoxin units per milliliter, involving the stepsof separating the red blood cells from said blood by centrifugation,mechanically degrading said red blood cells to release hemoglobin,clarifying said hemoglobin to remove cell debris, suspending saidhemoglobin in a pharmaceutically acceptable aqueous carrier medium tothereby form a hemoglobin solution, microporously filtering saidsolution to remove bacteria and red blood cell stroma, andultrafiltering said solution to remove phospholipids and to reduce theendotoxin level to less than about 0.5 endotoxin units permillimeter:the improvement comprising additionally chromatographicallytreating said hemoglobin solution in a high performance liquidchromatographic column by passing said solution over a chromatographicmedium for the ion-exchange affinity separation of endotoxin underconditions sufficient to produce a hemoglobin solution sufficientlypurified with respect to endotoxin so that the hemoglobin solution doesnot, upon in vivo administration to a mammal in a significant dosage,cause complement activation.
 32. In the preparation of an ultra purehemoglobin solution, from mammalian blood with an endotoxin level ofless than 7 endotoxin units per milliliter, involving the steps ofseparating the red blood cells from said blood by centrifugation,mechanically degrading said red blood cells to release hemoglobin,clarifying said hemoglobin to remove cell debris, suspending saidhemoglobin in a pharmaceutically acceptable aqueous carrier medium tothereby form a hemoglobin solution, microporously filtering saidsolution to remove bacteria and red blood cell stroma, andultrafiltering said solution to remove phospholipids and to reduce theendotoxin level to less than about 0.5 endotoxin units permillimeter:the improvement comprising additionally chromatographicallytreating said hemoglobin solution in a high performance liquidchromotographic column by passing said solution over a chromatographicmedium for the ion-exchange affinity separation of endotoxin underconditions sufficient to produce a hemoglobin solution having anendotoxin level of less than about 0.02 endotoxin units per milliliter(EU/ml).
 33. A pharmaceutical composition for transporting and releasingoxygen to living cells comprising a suspension of hemoglobin in apharmaceutically acceptable carrier, said suspension having an endotoxinlevel of less than about 0.02 EU/ml.
 34. A pharmaceutical composition ofclaim 33 wherein said hemoglobin comprises human hemoglobin.
 35. Apharmaceutical composition of claim 34 wherein said hemoglobin iscrosslinked.
 36. A pharmaceutical composition of claim 33 wherein saidhemoglobin comprises bovine hemoglobin.
 37. A pharmaceutical compositionof claim 36 wherein said hemoglobin is crosslinked.
 38. A method oftreating a mammal which has sustained a loss of blood by administeringto said mammal a therapeutically efficacious amount of a suspension ofhemoglobin in a pharmaceutically acceptable aqueous carrier, saidsuspension of hemoglobin having an endotoxin level of less than about0.5 EU/ml.
 39. A method of claim 38 wherein said suspension ofhemoglobin has an endotoxin level of less than about 0.02 EU/ml.
 40. Amethod of claim 37 wherein said hemoglobin comprises crosslinkedhemoglobin.
 41. A method of claim 40 wherein said hemoglobin compriseshuman hemoglobin.
 42. A method of claim 40 wherein said hemoglobincomprises bovine hemoglobin.
 43. A method of claim 40 wherein the mammaltreated is a human being.
 44. A method of claim 42 wherein the mammaltreated is a human being.
 45. A hemoglobin solution comprisinghemoglobin in an aqueous pharmaceutically-acceptable carrier medium,said hemoglobin solution having an endotoxin level of less than about0.5 endotoxin units per milliliter (EU/ml).
 46. A blood-substitute fortransporting and releasing oxygen to living cells comprisingcross-linked hemoglobin in an aqueous pharmaceutically-acceptablecarrier medium, said hemoglobin solution being substantially free of redblood cell stroma and having an endotoxin level of less than about 0.5endotoxin units per milliliter (EU/ml).
 47. A plasma-expander fortransporting and releasing oxygen to living cells comprisingcross-linked hemoglobin in an aqueous pharmaceutically-acceptablecarrier medium, said hemoglobin solution being substantially free of redblood cell stroma and having an endotoxin level of less than about 0.5endotoxin units per milliliter (EU/ml).
 48. A resuscitation fluid fortransporting and releasing oxygen to living cells comprisingcross-linked hemoglobin in an aqueous pharmaceutically-acceptablecarrier medium, said hemoglobin solution being substantially free of redblood cell stroma and having an endotoxin level of less than about 0.5endotoxin units per milliliter (EU/ml).